Methods of Treating Cancers Overexpressing Carm1 With EZH2 Inhibitors and Platinum-Based Antineoplastic Drugs

ABSTRACT

In some embodiments, therapeutic treatments for a disease such as a cancer arc disclosed, including pharmaceutical compositions and methods of using pharmaceutical compositions for treating the cancer wherein the cancer cells overexpress arginine methyltransferase CARM1. In some embodiments, the therapeutic treatments disclosed include methods comprising the step of administering a therapeutically effective dose of an enhancer of zeste homolog 2 (EZH2) inhibitor to a subject, including a human subject, wherein the cancer cells of the subject overexpress arginine methyltransferase CARM1. In some embodiments, the EZH2 inhibitors are administered in conjunction with platinum-based antineoplastic drugs.

GOVERNMENT SUPPORT

This invention was made with Government support under NIH Grant Nos.CA010815 and 5 R01 CA163377-04, awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

Therapeutic treatments of cancer based on overexpression of the argininemethyltransferase CARM1 gene are disclosed.

BACKGROUND OF THE INVENTION

Epithelial ovarian cancer (EOC) remains the most lethal gynecologicalmalignancy in the United States. Despite the recent advances in targetedtherapy in different types of cancer, platinum based therapies such ascisplatin remain the standard of care for EOC patients. Recentdiscoveries have demonstrated that ovarian cancer is composed ofmultiple separate diseases. High-grade serous ovarian cancer (HGSOC) isthe most common subtype (>70% of EOC cases) and accounts for themajority of EOC-associated mortalities. Although many patients initiallyrespond to cisplatin, the majority of these patients become resistantand the mechanism of this resistance is not fully understood. Epigeneticregulation has been shown to play an important role in tumor progressionand specifically in the development of platinum therapy resistance.Thus, it is important to identify epigenetic factors that areresponsible for cisplatin resistance in EOC.

SWI/SNF (Switch/Sucrose Non-Fermentable) is a chromatin remodelingcomplex that is often dysregulated in different types of cancer. Theactivity of the SWI/SNF complex is tightly regulated and multiplesubunits of SWI/SNF are post-translationally modified. BAF155, one ofthe subunits of SWI/SNF complex, is methylated by coactivator-associatedarginine methyltransferase 1 (CARM1), also known as protein argininemethyltransferase 4 (PRMT4). This modification has been shown to enhancetumor progression and metastasis. Wang, et al., Cancer Cell, 2014, 25,21-36. CARM1 has been shown to methylate substrates involved inepigenetic chromatin remodeling. This suggests that epigeneticmechanisms play a key role in CARM1-expressing cancers. Interestingly,the CARM1 gene is amplified in 10% of high-grade ovarian cancer patientsand CARM1 amplification is associated with poor prognosis in EOCpatients.

Polycomb-repressive complex 2 (PRC2) is a multiprotein complex thatnegatively regulates the expression of large numbers of genes bygenerating a silencing histone modification (H3K27me3) through itscatalytic subunit enhancer of zeste homolog 2 (EZH2). Many of the genesregulated by PRC2 are involved in cancer progression, and dysregulationof PRC2 function is observed in many different types of cancer includingEOC. Recent studies have shown that SWI/SNF and PRC2 complexes play anantagonistic role in tumorigenesis. Wilson, et al., Cancer Cell 2010,184, 316-328. EZH2 is highly expressed in many cancers, including breastcancer, prostate cancer, and lymphoma, and is frequently associated withtumor progression and poor outcomes. Furthermore, mutated forms of EZH2,including somatic heterozygous mutations of the Y641 and A677 residuesof the catalytic SET domain, are observed in some cancers includingdiffuse large B-cell lymphoma (DLBCL) and follicular lymphoma. Morin, etal., Nature 2011, 476, 298-303; Ryan, et al. PLoS ONE 2011, 6, e28585;Morin, et al., Nature Genet. 2010, 42, 181-185.

A number of selective small molecule EZH2 inhibitors have beenidentified and progressed into clinical development. Momparler and Côté,Expert Opin. Investig. Drugs 2015, 24, 1031-43; Melnick, Cancer Cell2012, 22, 569-70. EZH2 inhibitors are currently being investigated forthe treatment of cancers exhibiting overexpression of EZH2, including Bcell lymphomas such as DLBCL, Germinal center B-cell DLBCL (GCB-DLBCL),and non-Hodgkin's lymphoma, follicular lymphoma, multiple myeloma,INI1-negative tumors, synovial sarcoma, breast cancer, prostate cancer,and other solid tumors. For example, the EZH2 inhibitor tazemetostat(EPZ-6438) has potent activity against EZH2-mutated non-Hodgkin'slymphoma. Knutson, et al., Mol. Cancer. Ther. 2014, 13, 842-854.

The present invention provides the unexpected finding that EZH2inhibitors may be used to effectively treat cancers that overexpressCARM1. The present invention further provides the unexpected findingthat combinations of EZH2 inhibitors and platinum-based antineoplasticagents are surprisingly effective in the treatment of any of severalsubtypes of cancers, such as solid tumor cancers, wherein a biologicalsample exhibits CARM1 overexpression.

SUMMARY OF THE INVENTION

In an embodiment, the invention includes a method of treating a cancerin a human subject suffering from the cancer in which cancer cellsoverexpress arginine methyltransferase CARM1, the method comprising thestep of administering a therapeutically effective dose of an EZH2inhibitor to the human subject.

In an embodiment, the invention includes a method of treating a cancerin a human subject suffering from the cancer in which cancer cellsoverexpress arginine methyltransferase CARM1, the method comprising thestep of administering a therapeutically effective dose of an EZH2inhibitor to the human subject, wherein the cancer is ovarian cancer.

In an embodiment, the invention includes a method of treating a cancerin a human subject suffering from the cancer in which cancer cellsoverexpress arginine methyltransferase CARM1, the method comprising thestep of administering a therapeutically effective dose of an EZH2inhibitor to the human subject, wherein the cancer is epithelial ovariancancer.

In an embodiment, the invention includes a method of treating a cancerin a human subject suffering from the cancer in which cancer cellsoverexpress arginine methyltransferase CARM1, the method comprising thestep of administering a therapeutically effective dose of an EZH2inhibitor to the human subject, wherein the cancer is an epithelialovarian tumor.

In an embodiment, the invention includes a method of treating a cancerin a human subject suffering from the cancer in which cancer cellsoverexpress arginine methyltransferase CARM1, the method comprising thestep of administering a therapeutically effective dose of an EZH2inhibitor to the human subject, wherein the cancer is malignant ovariancancer.

In an embodiment, the invention includes a method of treating a cancerin a human subject suffering from the cancer in which cancer cellsoverexpress arginine methyltransferase CARM1, the method comprising thestep of administering a therapeutically effective dose of an EZH2inhibitor to the human subject, wherein the EZH2 inhibitor is selectedfrom the group consisting of(S)-1-(sec-butyl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-methyl-6-(6-(piperazin-1-yl)pyridin-3-yl)-1H-indole-4-carboxamide(GSK126):

tazemetostat:

(R,Z)-1-(1-(1-(ethylsulfonyl)piperidin-4-yl)ethyl)-N-((2-hydroxy-4-methoxy-6-methylpyridin-3-yl)methyl)-2-methyl-1H-indole-3-carbimidicacid (CPI-169):

1-cyclopentyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(4-(morpholinomethyl)phenyl)-1H-indazole-4-carboxamide(EPZ-5687):

N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl((1R,4R)-4-((2-methoxyethyl)(methyl)amino)cyclohexyl)amino)-2-methyl-5-(3-morpholinoprop-1-yn-1-yl)benzamide(EPZ-11989):

1-isopropyl-N-((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-6-(2-(4-methylpiperazin-1-yl)pyridin-4-yl)-1H-indazole-4-carboxamide(GSK343):

N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-3-methyl-6-(6-(4-methylpiperazin-1-yl)pyridin-3-yl)-1H-indole-4-carboxamide(GSK503):

1-isopropyl-6-(6-(4-isopropylpiperazin-1-yl)pyridin-3-yl)-N-((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-1H-indazole-4-carboxamide(UNC-1999):

6-cyano-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(pentan-3-yl)-1H-indole-4-carboxamide(E11):

(1S,2R,5R)-5-(4-amino-1H-imidazo[4,5-c]pyridin-1-yl)-3-(hydroxymethyl)-3-cyclopentene-1,2-diol(DZNep):

sinefungin:

and pharmaceutically acceptable salts, solvates, hydrates, cocrystals,or prodrugs thereof.

In an embodiment, the invention includes a method of treating a cancerin a human subject suffering from the cancer in which cancer cellsoverexpress arginine methyltransferase CARM1, the method comprising thestep of administering a therapeutically effective dose of an EZH2inhibitor to the human subject, wherein the overexpression of argininemethyltransferase CARM1 is at a level selected from the group of atleast 2%, at least 5%, at least 8%, at least 10%, and at least 15%relative to a level in normal epithelial cells.

In an embodiment, the invention includes a method of treating a cancerin a human subject suffering from the cancer in which cancer cellsoverexpress arginine methyltransferase CARM1, the method comprising thestep of administering a therapeutically effective dose of an EZH2inhibitor to the human subject, and further comprising the step ofadministering a therapeutically effective dose of a platinum drug to thehuman subject.

In an embodiment, the invention includes a method of treating a cancerin a human subject suffering from the cancer in which cancer cellsoverexpress arginine methyltransferase CARM1, the method comprising thestep of administering a therapeutically effective dose of an EZH2inhibitor to the human subject, and further comprising the step ofadministering a therapeutically effective dose of a platinum drug to thehuman subject, wherein the platinum drug is selected from the groupconsisting of cisplatin, carboplatin, oxaliplatin, satraplatin,picoplatin, nedaplatin, triplatin tetranitrate, lipoplatin (liposomalcisplatin), and pharmaceutically acceptable salts, solvates, hydrates,cocrystals, or prodrugs thereof.

In an embodiment, the invention includes a pharmaceutical compositionfor treating a cancer in a human subject suffering from the cancer inwhich cancer cells overexpress arginine methyl transferase CARM1, thepharmaceutical composition including a therapeutically effective amountof an enhancer of zeste homolog 2 (EZH2) inhibitor or a pharmaceuticallyacceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition includes a therapeutically effective amountof a platinum drug or a pharmaceutically acceptable salt, solvate,hydrate, cocrystal, or prodrug thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings.

FIG. 1 illustrates CARM1 expression in different types of cancer.

FIG. 2 illustrates overall survival in CARM1-expressing cells. Data wasobtained from the Cancer Genome Atlas.

FIG. 3 illustrates the co-occurrence of CARM1 amplification with themutations in HR DNA repair genes, BRCA1 and BRCA2.

FIG. 4 illustrates CARM1 expression in a panel of high-grade ovariancarcinoma.

FIG. 5 illustrates the results of a colony formation assay with a panelof normal and ovarian cancer cell lines in the presence of GSK126.

FIG. 6 illustrates a quantification of the data shown in FIG. 5.

FIG. 7 illustrates GSK126 response curves in a panel of normal andovarian cancer cell lines. Error bars indicate standard error (S.E.) forn=3. *, p<0.05.

FIG. 8 illustrates IC₅₀ results for the panel of normal and ovariancancer cell lines.

FIG. 9 illustrates a Western blot of CARM1 and BAF155me2 in CARM1depleted cells with a control (Actin).

FIG. 10 illustrates the results of a colony formation assay (unstained,magnified) with normal and CARM1-depleted cells, in DMSO (control) andtreated with GSK126.

FIG. 11 illustrates the results of a colony formation assay with normaland CARM1-depleted cells, in DMSO (control) and treated with GSK126.

FIG. 12 illustrates a quantification of the data shown in FIG. 11.

FIG. 13 illustrates the results of treatment of CARM1 depleted cellswith GSK126. Error bars indicate S.E. for n=3. *, p<0.05.

FIG. 14 illustrates CARM1 amplification and BRCA1/2 mutation profiles inthe TCGA HGSOC database.

FIG. 15 illustrates relative expression of CARM1 in laser capture andmicrodissected (LCM) high-grade serous ovarian cancer (HGSOC) and normalhuman ovarian surface epithelial (HOSE) cells.

FIG. 16 illustrates relative expression of CARM1 in laser captured andmicrodissected HGSOC and fallopian tube epithelial (FTE) cells.

FIG. 17 illustrates expression of CARM1 and EZH2 in the indicated EOCcells lines, HOSE, and FTE cells determined by immunoblot. Expression ofβ-actin was used as a loading control.

FIG. 18 illustrates overall survival of EOC patients with or withoutCARM1 amplification based on TCGA copy number analysis (n=477).

FIG. 19 illustrates overall survival of EOC patients with high or lowCARM1 expression in an EOC microarray database. The cutoff expressionlevel was statistically determined by a continuous expression model.

FIG. 20 illustrates expression of CARM1 and a loading control β-actin inCARM1 and a loading control β-actin in CARM1 high parental andCRISPR-mediated CARM1 knockout (KO) A1847 cells.

FIG. 21 illustrates growth curves of parental control and CARM1 knockoutA1847 cells. Mean of three independent experiments with SEM.

FIG. 22 illustrates the expression of CARM1 in A1847 cells expressingthe indicated shCARM1 or controls.

FIG. 23 illustrates growth curves of control and CARM1 knockdown A1847cells. Mean of three independent experiments with standard deviation.

FIG. 24 illustrates the expression of CARM1 in high OVCAR10 EOC cellsexpressing the indicated shCARM1 or controls.

FIG. 25 illustrates growth curves of control and CARM1 knockdown highOVCAR10 EOC cells. Mean of three independent experiments with standarddeviation.

FIG. 26 illustrates that CARM1-expressing cells are selectivelysensitized to EZH2 inhibitors. Equal number of parental control or CARM1knockout A1847 cells were plated and treated with each of the 23individual epigenetic inhibitors for 14 days. The media with theinhibitors was refreshed every 3 days. Cell growth was quantified asintegrated density using NIH Image J software. Quantification of theaverage integrated density graphed as a scatter plot. The X-axisindicates the relative growth of treated CARM1 high parental A1847 cellscompared with DMSO vehicle controls. Y-axis indicates the relativegrowth of treated CARM1 knockout A1847 cells compared with DMSO vehiclecontrols. n=4; and error bars represents SEM.

FIG. 27 illustrates a table of EZH2 inhibitors that are selectiveagainst CARM1 expression. CARM1-high parental controls and CARM1knockout A1847 cells were treated with the indicated concentration ofthe small molecules in a 14-day colony formation assay. Integrateddensity for each well was calculated using NIH Image J software. Therelative growth of the indicated cells was determined by normalizing tothe vehicle control treated cells. n=4 and p-value was calculated usinga two-tailed t-test.

FIG. 28 illustrates representative images of colonies formed by thecells treated with the indicated inhibitors. GSK126 and UNC1999representative positive hits from the screen. Note that CI994 was used anegative control that showed no difference between parental and CARM1knockout cells.

FIG. 29 illustrates the expression of CARM1, H3K27Me3, EZH2, and H3 inparental control and CARM1 knockout A1847 cells.

FIG. 30 illustrates relative expression of EZH2 in human ovarian surfaceepithelial (HOSE, n=10) cells and laser capture-microdissectedhigh-grade serous ovarian carcinoma (LCM HGSOC, n=35) with high CARM1expression.

FIG. 31 illustrates relative expression of EZH2 in fallopian tubeepithelial cells (n=24) and LCM HGSOC (n=10) with high CARM1 expression.

FIG. 32 illustrates that the EZH2 inhibitor GSK126 decreases H3K27Me3levels in a dose-dependent manner. A1847 cells were treated with theindicated concentrations of GSK126 for 72 hours and examined forexpression of the indicated proteins by immunoblot. Expression ofhistone H3 and β-actin was used as a loading control.

FIG. 33 illustrates relative growth of the indicated HOSE, FTE, and EOCcancer cell lines with high or low CARM1 expression treated with 10 μMGSK126 or vehicle in a colony formation assay as determined by NIH ImageJ quantification.

FIG. 34 illustrates expression of H3K27Me3 in the indicated EOC celllines with high or low CARM1 expression treated with or without 10 μMGSK126. Expression of β-actin was used as a loading control.

FIG. 35 illustrates representative images of acini formed by indicatedcells treated with or without 10 μM GSK126 in 3D cultures using Matrigelextracelluar matrix for 12 days. Scale Bars=50 of measurable units (AU)using the NIH Image J Software.

FIG. 36 illustrates the quantification of the diameter of acini formedby the indicated cells with or without 10 μM GSK126 treatment in 3Dculture for 12 days. (N=30 acini per sample).

FIG. 37 illustrates control parental and CARM1 knockout A1847 cells weretreated with 10 μM GSK126 or vehicle for 7 days. Percentage of Annexin Vpositive apoptotic cells was quantified. *p<0.001 and #p>0.05.

FIG. 38 illustrates levels of cleaved caspase 3 and cleaved PARP incontrol parental and CARM1 knockout A1847 cells treated with 10 μMGSK126 for 7 days. Expression of β-actin was used as a loading control.

FIG. 39A illustrates the expression of apoptosis markers cleaved inCARM1 high OVCAR10 cells as a result of treatment with GSK126. CARM1high OVCAR10 cells were treated with 10 μM GSK126 or vehicle control for7 days. The expression of apoptosis markers cleaved caspase 3 andcleaved PARP were determined by immunoblot. Expression of β-actin wasused as a loading control.

FIG. 39B illustrates that EZH2 inhibitor GSK126 did not affect PRC2subunits EZH2 and SUZ12 interaction in CARM1 high A1847 cells. A1847cells were treated with 10 μM GSK126 or vehicle control for 7 days. Thecells were subjected to immunoprecipitation (IP) analysis by using ananti-EZH2 antibody. The immunoprecipitated product was examined for EZH2and SUZ12 expression by immunoblot. An isotype-matched IgG was used as acontrol.

FIGS. 40 to 43 illustrate CARM1-high A1847 cells were infected with alentivirus encoding shEZH2 targeting the 3′ untranslated region (UTR) ofthe human EZH2 gene together with a retrovirus encoding wild-type EZH2(WT) or a SET domain-deleted EZH2 mutant (EZH2 ASET). Drug-selectedcells were examined for EZH2, H3K27Me3, apoptosis markers cleavedcaspase 3 and cleaved PARP p85, and loading control (β-actin) byimmunoblot (FIG. 40), quantified for Annexin positive apoptotic cells byFACS (FIG. 41), subjected to a colony formation assay (FIG. 42), andquantified for the relative cell growth based on colony formation usingNIH Image J (FIG. 43). Means of three independent experiments with SEMare shown.

FIG. 44 illustrates an experimental strategy used to identifyCARM1-regulated, clinically relevant EZH2/H3K27Me3 target genes.Ingenuity Pathway Analysis revealed that the top pathway enriched forthe identified CARM1-regulated EZH2/H3K27Me3 target genes was apoptosis.Specifically, 19 of the 36 identified genes (including DAB2, DLC1, andNOXA) are known to promote apoptosis (the full list is available in FIG.49).

FIG. 45 illustrates examples of EZH2 and H3K27Me3 ChIP-seq and RNA-seqtracks of the newly identified CARM1-regulated EZH2/H3K27Me3 targetgenes in the indicated parental control and CARM1 knockout A1847 cells.

FIGS. 46 to 48 illustrate that EZH2/H3K27Me3 target genes are enrichedin genes upregulated by CARM1 knockout in A1847 cells. FIGS. 46 and 47illustrate ChIP-seq peaks compilations of EZH2 (FIG. 46) and H3K27Me3(FIG. 47) relative to transcription starting site (TSS) of the 218direct EZH2/H3K27Me3 target genes that are upregulated in CARM1 knockoutcells compared with control CARM1-high A1847 cells. FIG. 48 illustratesthat EZH2/H3K27Me3 direct target genes are enriched in genes upregulatedby CARM1 knockout.

FIG. 49 illustrates a table that lists 19 identified EZH2/H3K27Me3target apoptosis-promoting genes that are upregulated by CARM1 knockoutand negatively correlate with CARM1 expression in the 579 TCGA highgrade serous ovarian carcinoma database.

FIG. 50 illustrates differentially expressed genes in control parentaland CARM1 knockout A1847 cells identified by RNA-seq (>3-fold) weresubjected to Ingenuity Pathway Analysis for upstream regulators. Theanalysis revealed that SMARCA4 (also known as BRG1, a catalytic subunitof the SWI/SNF complex) was the top upstream regulator of thesedifferentially expressed genes.

FIG. 51 illustrates a table that lists the top 10 upstream transcriptionfactors that are enriched by the differentially expressed genes incontrol parental and CARM1 knockout A1847 cells determined by IngenuityPathway Analysis.

FIG. 52 illustrates the expression of CARM1, BAF155, and BAF155Me incontrol parental and CARM1 knockout A1847 cells. Expression of β-actinwas used as a loading control.

FIGS. 53 and 54 illustrate that CARM1 regulates BAF155Me and EZH2inhibition did not affect BAF155Me levels. FIG. 53 illustrates that theindicated CARM1-high cells were infected with the indicated shCARM1 orcontrols. Expression of CARM1 and BAF155Me levels and loading control(β-actin) was determined by immunoblot. FIG. 54 illustrates that theindicated cells were treated with 10 μM GSK126 or vehicle control for 7days. Expression of BAF155 and a loading control (β-actin) wasdetermined by immunoblot.

FIG. 55 illustrates the relative expression of DAB2, DLC1, and NOXA inparental control and CARM1 knockout A1847 cells that were treated with10 μM GSK126 or vehicle controls. mRNA expression of the indicated geneswas determined by qRT-PCR. *p<0.002 compared with parental controls.

FIG. 56 illustrates the relative expression of DAB2, DLC1, and NOXA inCARM1 high OVCAR10 cells treated with 10 μM GSK126 or vehicle controlfor 7 days. Expression of the indicated BAF155/EZH2 target genes wasdetermined by qRT-PCR. *p<0.001. Mean of three independent experimentswith SEM.

FIG. 57 illustrates the relative mRNA expression of BAF155Me target geneTIMP3, which was determined by qRT-PCR. Parental control and CARM1knockout A1847 cells were treated with 10 μM GSK126 or vehicle controls.*p<0.001. Mean of three independent experiments with SEM.

FIGS. 58 to 61 illustrate the results of a ChIP analysis using anti-EZH2(FIG. 58), anti-H3K27Me3 (FIG. 59), anti-BAF155 (FIG. 60), or anti-RNAPol II (FIG. 61) antibodies. An isotype matched IgG was used as anegative control. ChIP products were subjected to qPCR analysis usingprimers specific for the promoter regions of the human DAB2, DLC1, andNOXA genes. Data is representative of three independent experiments. *p<0.001 compared with IgG controls and #p<0.001 compared with parentalcontrols.

FIGS. 62 to 65 illustrate the results of a ChIP analysis in parentalcontrol and CARM1 knockout A1847 cells treated with 10 μM GSK126 orvehicle controls. The cells were subjected to ChIP analysis usinganti-EZH2 (FIG. 62), anti-H3K27Me3 (FIG. 63), anti-BAF155 (FIG. 64), oranti-RNA Pol II (FIG. 65) antibodies. An isotype-matched IgG was used asa negative control. ChIP products were subjected to qPCR analysis usingprimers specific for the promoter regions of the human TIMP3 genes. Meanof three independent experiments with SEM. *p<0.001 compared withcontrols.

FIG. 66 illustrates a model proposed for the molecular basis of theCARM1-dependent effects of EZH2 inhibition. Without being limited to anyone theory, CARM1 regulates the antagonism between the BAF155-containingSWI/SNF complex and the EZH2-containing PRC2 complex by methylatingBAF155. This correlates with the silencing of the tumor-suppressiveBAF155/EZH2 target genes due to displacement of the methylated BAF155 byEZH2. Expression of these genes can be restored by EZH2 inhibition.Error bars represent SEM p-values are from two-tailed t-test.

FIGS. 67 to 71 illustrate that CARM1-mediated R1064 BAF155 methylationdisplaces BAF155 with EZH2 at the promoters of EZH2/BAF155 target genes.FIG. 67 illustrates CARM1-expressing A1847 cells that were infected witha lentivirus encoding shBAF155 targeting the 3′ untranslated region(UTR) of the human BAF155 gene together with a retrovirus encodingwild-type BAF155 (WT) or a BAF155 R1064K mutant. Expression of BAF155and BAF155Me was determined by immunoblot. B-actin expression was usedas a loading control. FIG. 68 illustrates the expression of theindicated BAF155/EZH2 target genes by qRT-PCT. FIGS. 69 to 71 illustratethe results of a ChIP analysis using anti-EZH2 (FIG. 69), anti-H3K27Me3(FIG. 70), or anti-BAF155 (FIG. 71) antibodies, where isotype-matchedIgG was used as a negative control. ChIP products were subjected to qPCRanalysis using primers specific for the promoter regions of the humanDAB2, DLC1, and NOXA genes. Error bars represent SEM. P-values are froma two-tailed t-test.

FIGS. 72A and 72B illustrate that CARM1 promotes the expression ofBAF155Me target genes. FIG. 72A illustrates the expression of theBAF155Me target gene TIMP3. CARM1-expressing A1847 cells were infectedwith a lentivirus encoding shBAF155 targeting the 3′ untranslated region(UTR) of the human BAF155 gene together with a retrovirus encodingwild-type BAF155 (WT) or a BAF155 R1064K mutant. Expression of BAF155Metarget gene TIMP3 was determined by qRT-PCR in the indicated cells. Meanof three independent experiments with SEM. *p<0.001. FIG. 72Billustrates the results of a ChIP analysis using an anti-BAF155 antibodyin CARM1-expressing A1847 cells. An isotype matched IgG was used as anegative control. ChIP products were subjected to qPCR analysis usingprimers specific for the promoter of the human TIMP3 gene. Error barsrepresent SEM. *p<0.001.

FIGS. 73 to 75 illustrate that EZH2 inhibition suppressed the growth ofCARM1-expressing ovarian tumors in vivo. FIG. 73 illustrates images oftumors dissected from control or GSK126 treated mice. CARM1-high A1847cells were injected subcutaneously into immunocompromised NSG mice(n=5/group). Tumors were allowed to establish for one week before themice were randomized into two different treatment groups. Mice weretreated with vehicle control or GSK126 (50 mg/kg daily) for anadditional three weeks. At the end of treatment, the mice wereeuthanized. Bar=1 cm. FIG. 74 illustrates a difference in tumor sizemeasured as a surrogate for tumor burden from the control and GSK126treated mice at the indicated time point. FIG. 75 illustrates adifference in tumor weight measured as a surrogate for tumor burden fromcontrol and GSK126-treated mice. The mice were injected with CARM1knockout A1847 cells.

FIG. 76 illustrates images of reproductive tracks with tumors fromcontrol or GSK126 treated mice. CARM1-high A1847 ovarian cancer cellswere unilaterally injected into the ovarian bursa sac ofimmunocompromised mice (n=5/group). Tumors were allowed to establish forone week before the mice were randomized into two different treatmentgroups. Mice were treated with vehicle control or GSK126 (50 mg/kgdaily) for an additional three weeks. At the end of treatment, the micewere euthanized.

FIG. 77 illustrates tumor weights for the tumors described in FIG. 76.Tumor weight was measured as a surrogate for tumor burden from thecontrol and GSK126 treated mice.

FIG. 78 illustrates the percent survival for the animals treated in FIG.76. After stopping treatment, the mice from the indicated groups werefollowed for survival. Shown in FIG. 78 are the Kaplan-Meier survivalcurves for GSK126 or vehicle treated mice. P-value was calculated bylog-rank test.

FIG. 79 illustrates immunohistochemical analysis for serial sections oftumors dissected from the indicated treatment groups where the tumorsections were stained for H3K27Me3, EZH2, cleaved caspase 3, and Ki67.Scale bar=50 μm.

FIG. 80 illustrates histological scoring (H-score) of the indicatedproteins that was calculated for 3 separate fields from 5 tumors from 5individual mice from each of the indicated groups.

FIG. 81 illustrates the expression of the indicated EZH2/BAF155 targetgenes that was determined by qRT-PCR in the tumors dissected from theindicated treatment groups.

FIGS. 82 to 84 illustrate immunohistochemical analysis for serialsections of tumors dissected from mice injected with CARM1 knockoutA1847 cells. Serial sections of tumors dissected from the indicatedtreatment groups described in FIGS. 73 to 75 were subjected toimmunohistochemical staining for H3K27Me3, EZH2, cleaved caspase 3, andKi67 (FIG. 82). Scale bar=50 μm. Histological score (H-score) of theindicated proteins was calculated for 3 separate fields from 5 tumorsfrom 5 individual mice from each of the indicated groups (FIG. 83).Expression of the indicated EZH2/BAF155 target genes was determined byqRT-PCR in the tumors dissected from the indicated treatment groups(FIG. 84).

FIG. 85 illustrates how CARM1 regulates the antagonism between SWI/SNFand PRC2 through methylating BAF155. EZH2 inhibition reactivates thesetumor suppressive genes to promote apoptosis, inhibit proliferation, andsuppress tumor growth.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference in their entireties.

Definitions

The terms “co-administration,” “co-administering,” “administered incombination with,” “administering in combination with,” “simultaneous,”and “concurrent,” as used herein, encompass administration of two ormore active pharmaceutical ingredients to a human subject so that bothactive pharmaceutical ingredients and/or their metabolites are presentin the human subject at the same time. Co-administration includessimultaneous administration in separate compositions, administration atdifferent times in separate compositions, or administration in acomposition in which two or more active pharmaceutical ingredients arepresent. Simultaneous administration in separate compositions andadministration in a composition in which both agents are present is alsoencompassed in the methods of the invention.

The terms “active pharmaceutical ingredient” and “drug” include EZH2inhibitors and platinum-based antineoplastic drugs.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound or combination of compounds as describedherein that is sufficient to effect the intended application including,but not limited to, disease treatment. A therapeutically effectiveamount may vary depending upon the intended application (in vitro or invivo), or the human subject and disease condition being treated (e.g.,the weight, age and gender of the subject), the severity of the diseasecondition, the manner of administration, etc. which can readily bedetermined by one of ordinary skill in the art. The term also applies toa dose that will induce a particular response in target cells (e.g., thereduction of platelet adhesion and/or cell migration). The specific dosewill vary depending on the particular compounds chosen, the dosingregimen to be followed, whether the compound is administered incombination with other compounds, timing of administration, the tissueto which it is administered, and the physical delivery system in whichthe compound is carried.

A “therapeutic effect” as that term is used herein, encompasses atherapeutic benefit and/or a prophylactic benefit in a human subject. Aprophylactic effect includes delaying or eliminating the appearance of adisease or condition, delaying or eliminating the onset of symptoms of adisease or condition, slowing, halting, or reversing the progression ofa disease or condition, or any combination thereof.

The term “pharmaceutically acceptable salt” refers to salts derived froma variety of organic and inorganic counter ions known in the art.Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids. Inorganic acids from which salts canbe derived include, for example, hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid and phosphoric acid. Organic acids from whichsalts can be derived include, for example, acetic acid, propionic acid,glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid and salicylic acid. Pharmaceutically acceptablebase addition salts can be formed with inorganic and organic bases.Inorganic bases from which salts can be derived include, for example,sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese and aluminum. Organic bases from which salts can bederived include, for example, primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins. Specific examples includeisopropyl amine, trimethylamine, diethylamine, triethylamine,tripropylamine, and ethanolamine. In some embodiments, thepharmaceutically acceptable base addition salt is chosen from ammonium,potassium, sodium, calcium, and magnesium salts. The term “cocrystal”refers to a molecular complex derived from a number of cocrystalformers. Unlike a salt, a cocrystal typically does not involve hydrogentransfer between the cocrystal and the drug, and instead involvesintermolecular interactions, such as hydrogen bonding, aromatic ringstacking, or dispersive forces, between the cocrystal former and thedrug in the crystal structure.

“Pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” is intended to include any and all solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and inert ingredients. The use of suchpharmaceutically acceptable carriers or pharmaceutically acceptableexcipients for active pharmaceutical ingredients is well known in theart. Except insofar as any conventional pharmaceutically acceptablecarrier or pharmaceutically acceptable excipient is incompatible withthe active pharmaceutical ingredient, its use in the therapeuticcompositions of the invention is contemplated. Additional activepharmaceutical ingredients, such as other drugs, can also beincorporated into the described compositions and methods.

“Prodrug” is intended to describe a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound described herein. Thus, the term “prodrug” refers to aprecursor of a biologically active compound that is pharmaceuticallyacceptable. A prodrug may be inactive when administered to a subject,but is converted in vivo to an active compound, for example, byhydrolysis. The prodrug compound often offers the advantages ofsolubility, tissue compatibility or delayed release in a mammalianorganism (see, e.g., Bundgaard, Design of Prodrugs, Elsevier, Amsterdam,1985). The term “prodrug” is also intended to include any covalentlybonded carriers, which release the active compound in vivo whenadministered to a subject. Prodrugs of an active compound, as describedherein, may be prepared by modifying functional groups present in theactive compound in such a way that the modifications are cleaved, eitherin routine manipulation or in vivo, to yield the active parent compound.Prodrugs include, for example, compounds wherein a hydroxy, amino ormercapto group is bonded to any group that, when the prodrug of theactive compound is administered to a mammalian subject, cleaves to forma free hydroxy, free amino or free mercapto group, respectively.Examples of prodrugs include, but are not limited to, acetates, formatesand benzoate derivatives of an alcohol, various ester derivatives of acarboxylic acid, or acetamide, formamide and benzamide derivatives of anamine functional group in the active compound.

Unless otherwise stated, the chemical structures depicted herein areintended to include compounds which differ only in the presence of oneor more isotopically enriched atoms. For example, compounds where one ormore hydrogen atoms is replaced by deuterium or tritium, or wherein oneor more carbon atoms is replaced by ¹³C- or ¹⁴C-enriched carbons, arewithin the scope of this invention.

When ranges are used herein to describe, for example, physical orchemical properties such as molecular weight or chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included. Use of the term “about” whenreferring to a number or a numerical range means that the number ornumerical range referred to is an approximation within experimentalvariability (or within statistical experimental error), and thus thenumber or numerical range may vary. The variation is typically from 0%to 15%, from 0% to 10%, from 0% to 5% of the stated number or numericalrange. The term “comprising” (and related terms such as “comprise” or“comprises” or “having” or “including”) includes those embodiments suchas, for example, an embodiment of any composition of matter, method orprocess that “consist of” or “consist essentially of” the describedfeatures.

Compounds used in the methods of the invention also include crystallineand amorphous forms of those compounds, including, for example,polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs(including anhydrates), conformational polymorphs, and amorphous formsof the compounds, as well as mixtures thereof. “Crystalline form” and“polymorph” are intended to include all crystalline and amorphous formsof the compound, including, for example, polymorphs, pseudopolymorphs,solvates, hydrates, unsolvated polymorphs (including anhydrates),conformational polymorphs, and amorphous forms, as well as mixturesthereof, unless a particular crystalline or amorphous form is referredto.

Methods of Treating Cancers and Other Diseases

The compositions and methods described herein can be used in a methodfor treating diseases. In an embodiment, they are for use in treatinghyperproliferative disorders. They may also be used in treating otherdisorders as described herein and in the following paragraphs.

In some embodiments, the hyperproliferative disorder is cancer. Inselected embodiments, the cancer is selected from the group consistingof ovarian cancer, epithelial ovarian cancer, non-Hodgkin's lymphomas(such as diffuse large B-cell lymphoma), acute myeloid leukemia, thymuscancer, brain cancer, lung cancer, squamous cell cancer, skin cancer,eye cancer, retinoblastoma, intraocular melanoma, oral cavity andoropharyngeal cancer, bladder cancer, gastric cancer, stomach cancer,pancreatic cancer, breast cancer, cervical cancer, head and neck cancer,renal cancer, kidney cancer, liver cancer, prostate, colorectal cancer,bone (e.g., metastatic bone), esophageal cancer, testicular cancer,gynecological cancer, thyroid cancer, central nervous system lymphomas,AIDS-related cancers (e.g. lymphoma and Kaposi's sarcoma), viral-inducedcancers such as cervical carcinoma (human papillomavirus), B-celllymphoproliferative disease and nasopharyngeal carcinoma (Epstein-Barrvirus), Kaposi's sarcoma and primary effusion lymphomas, hepatocellularcarcinoma (hepatitis B and hepatitis C viruses), and T-cell leukemias(human T-cell leukemia virus-1), B cell acute lymphoblastic leukemia,Burkitt's leukemia, juvenile myelomonocytic leukemia, hairy cellleukemia, Hodgkin's disease, multiple myeloma, mast cell leukemia, andmastocytosis.

In an embodiment, the hyperproliferative disorder is EZH2-mutatedcancer. EZH2-mutated cancers are described, e.g., in InternationalPatent Application Publication No. WO 2015/128837 A1 and U.S. PatentApplication Publication No. 2016/0361309, the disclosures of which areincorporated by reference herein. EZH2-mutated cancers include pointmutations, such as the alanine-to-valine mutation at residue 687 of EZH2(the A687V mutation).

Efficacy of the compounds and combinations of compounds described hereinin treating, preventing and/or managing the indicated diseases ordisorders can be tested using various models known in the art, whichprovide guidance for treatment of human disease. For example, models fordetermining efficacy of treatments for ovarian cancer are described,e.g., in Mullany, et al., Endocrinology 2012, 153, 1585-92; and Fong, etal., J. Ovarian Res. 2009, 2, 12. Models for determining efficacy oftreatments for pancreatic cancer are described in Herreros-Villanueva,et al., World J. Gastroenterol. 2012, 18, 1286-1294. Models fordetermining efficacy of treatments for breast cancer are described,e.g., in Fantozzi, Breast Cancer Res. 2006, 8, 212. Models fordetermining efficacy of treatments for melanoma are described, e.g., inDamsky, et al., Pigment Cell & Melanoma Res. 2010, 23, 853-859. Modelsfor determining efficacy of treatments for lung cancer are described,e.g., in Meuwissen, et al., Genes & Development, 2005, 19, 643-664.Models for determining efficacy of treatments for lung cancer aredescribed, e.g., in Kim, Clin. Exp. Otorhinolaryngol. 2009, 2, 55-60;and Sano, Head Neck Oncol. 2009, 1, 32. Models for determining efficacyin B cell lymphomas, such as diffuse large B cell lymphoma (DLBCL),include the PiBCL1 murine model with BALB/c (haplotype H-2d) mice.Illidge, et al., Cancer Mother. & Radiopharm. 2000, 15, 571-80. Efficacyof treatments for Non-Hodgkin's lymphoma may be assessed using the 38C13murine model with C3H/HeN (haplotype 2-Hk) mice or alternatively the38C13 Her2/neu model. Timmerman, et al., Blood 2001, 97, 1370-77;Penichet, et al., Cancer Immunolog. Immunother. 2000, 49, 649-662.Efficacy of treatments for chronic lymphocytic leukemia (CLL) may beassessed using the BCL1 model using BALB/c (haplotype H-2d) mice. Dutt,et al., Blood 2011, 117, 3230-29.

EZH2 Inhibitors

In an embodiment, the invention includes a method of treating a cancerin a human subject suffering from the cancer in which cancer cellsoverexpress arginine methyltransferase CARM1, the method comprising thestep of administering a therapeutically effective dose of an activepharmaceutical ingredient that is an EZH2 inhibitor to the humansubject. In an embodiment, the foregoing method further comprises thestep of administering a therapeutically effective dose of another activepharmaceutical ingredient, such as a platinum-based drug. The EZH2inhibitor may be any EZH2 inhibitor known in the art. Suitable EZH2inhibitors are described, for example, in Momparler and Côté, ExpertOpin. Investig. Drugs 2015, 24, 1031-43. In particular, the EZH2inhibitor is an EZH2 inhibitor described in more detail in the followingparagraphs.

In an embodiment, the EZH2 inhibitor is(S)-1-(sec-butyl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-methyl-6-(6-(piperazin-1-yl)pyridin-3-yl)-1H-indole-4-carboxamide,also known as GSK2816126 or GSK126 (Formula (1)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal,tautomer, or prodrug thereof. GSK126 is commercially available frommultiple suppliers. The synthesis and properties of GSK126 and othersuitable EZH2 inhibitors are described in, e.g., U.S. Pat. Nos.8,536,179, 8,846,935, and 8,637,509, the disclosures of which areincorporated by reference herein.

In an embodiment, the EZH2 inhibitor isN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-3-methyl-6-(6-(4-methylpiperazin-1-yl)pyridin-3-yl)-1H-indole-4-carboxamide,also known as GSK503 (Formula (2)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal,tautomer, or prodrug thereof. The synthesis and properties of Formula(2) are described in, e.g., U.S. Pat. Nos. 8,536,179, 8,846,935, and8,637,509, the disclosures of which are incorporated by referenceherein.

In an embodiment, the EZH2 inhibitor is1-isopropyl-N-((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-6-(2-(4-methylpiperazin-1-yl)pyridin-4-yl)-1H-indazole-4-carboxamide,also known as GSK343 (Formula (3)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal,tautomer, or prodrug thereof. The synthesis and properties of Formula(3), and other EZH2 inhibitors suitable for use with the presentmethods, are described in, e.g., U.S. Pat. Nos. 8,637,509, 8,846,935,and 9,018,382, the disclosures of which are incorporated by referenceherein.

In an embodiment, the EZH2 inhibitor isN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide,also known as tazemetostat or EPZ-6438 (Formula (4)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal,tautomer, or prodrug thereof. Tazemetostat is commercially availablefrom Epizyme, Inc., and is described in Knutson, et al., Mol. CancerTher. 2014, 13, 842-54. The synthesis and properties of tazemetostat andother suitable EZH2 inhibitors are described in, e.g., U.S. Pat. Nos.8,765,732, 8,410,088, and 9,090,562, the disclosures of which areincorporated by reference herein.

In an embodiment, the EZH2 inhibitor is(R,Z)-1-(1-(1-(ethylsulfonyl)piperidin-4-yl)ethyl)-N-((2-hydroxy-4-methoxy-6-methylpyridin-3-yl)methyl)-2-methyl-1H-indole-3-carbimidicacid, also known as CPI-169 (Formula (5)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal,tautomer, or prodrug thereof. The synthesis and properties of Formula(5) and other suitable EZH2 inhibitors are described in, e.g., U.S.Patent Application Publication No. US 2016/0009718 A1, the disclosuresof which are incorporated by reference herein.

In an embodiment, the EZH2 inhibitor is1-cyclopentyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(4-(morpholinomethyl)phenyl)-1H-indazole-4-carboxamide,also known as EPZ-5687 (Formula (6)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal,tautomer, or prodrug thereof.

In an embodiment, the EZH2 inhibitor isN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl((1R,4R)-4-((2-methoxyethyl)(methyl)amino)cyclohexyl)amino)-2-methyl-5-(3-morpholinoprop-1-yn-1-yl)benzamide,also known as EPZ-11989 (Formula (7)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal,tautomer, or prodrug thereof.

In an embodiment, the EZH2 inhibitor is1-isopropyl-6-(6-(4-isopropylpiperazin-1-yl)pyridin-3-yl)-N-((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-1H-indazole-4-carboxamide,also known as UNC-1999 (Formula (8)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal,tautomer, or prodrug thereof.

In an embodiment, the EZH2 inhibitor is6-cyano-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(pentan-3-yl)-1H-indole-4-carboxamide,also known as E11 (Formula (9)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal,tautomer, or prodrug thereof. The synthesis and properties of E11 aredescribed in, e.g., Qi, et al., Proc. Natl. Acad. Sci. USA 2012, 109,21360-65.

In an embodiment, the EZH2 inhibitor is(1S,2R,5R)-5-(4-amino-1H-imidazo[4,5-c]pyridin-1-yl)-3-(hydroxymethyl)-3-cyclopentene-1,2-diol,also known as DZNep (Formula (10)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal,tautomer, or prodrug thereof.

In an embodiment, the EZH2 inhibitor is(2S,5S)-2,5-diamino-6-((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)hexanoicacid, also known as 5′-deoxy-5′-(1,4-diamino-4-carboxybutyl)adenosineand sinefungin (Formula (11)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal,tautomer, or prodrug thereof. The isolation of sinefungin is described,e.g., in U.S. Pat. No. 3,758,681, the disclosure of which isincorporated by reference herein. The synthesis of sinefungin isdescribed, e.g., in Maguire, et al., J. Org. Chem. 1990, 55, 948. In anembodiment, the EZH2 inhibitor is a derivative of sinefungin. Sinefunginderivatives, and are described, e.g., in French Patent No. FR 2664277 B1and in Zheng, et al., J. Am. Chem. Soc. 2012, 134, 18004-14, thedisclosures of which are incorporated by reference herein.

In an embodiment, the EZH2 inhibitor is CPI-1205, or a pharmaceuticallyacceptable salt, solvate, hydrate, cocrystal, tautomer, or prodrugthereof. CPI-1205 is available from Constellation Pharmaceuticals.

In some embodiments, the EZH2 inhibitor is GSK126, UNC1999, or acombination combination thereof, or a pharmaceutically acceptable salt,solvate, hydrate, cocrystal, tautomer, or prodrug thereof.

In some embodiments, the EZH2 inhibitor is GSK126 or a pharmaceuticallyacceptable salt, solvate, hydrate, cocrystal, tautomer, or prodrugthereof.

In some embodiments, the EZH2 inhibitor is UNC1999 or a pharmaceuticallyacceptable salt, solvate, hydrate, cocrystal, tautomer, or prodrugthereof.

Platinum-Based Antineoplastic Drugs

In an embodiment, the invention includes a method of treating a cancerin a human subject suffering from the cancer in which cancer cellsoverexpress arginine methyltransferase CARM1, the method comprising thestep of administering a therapeutically effective dose of an activepharmaceutical ingredient that is an EZH2 inhibitor to the humansubject, and further comprising the step of administering an activepharmaceutical ingredient that is a platinum-based antineoplastic drug.The platinum-based antineoplastic drug may be administered before,concurrently with, or after the EZH2 inhibitor. Any platinum-basedantineoplastic drug known in the art may be used, as described, e.g., inKelland, Nature Rev. Cancer 2007, 7, 573-84. In particular, it is one ofthe platinum-based antineoplastic drugs described in more detail in thefollowing paragraphs.

In an embodiment, the platinum-based antineoplastic drug is selectedfrom the group consisting of cisplatin, carboplatin, oxaliplatin,satraplatin, picoplatin, nedaplatin, triplatin tetranitrate, lipoplatin(liposomal cisplatin), combinations thereof, and pharmaceuticallyacceptable salts, solvates, hydrates, cocrystals, or prodrugs thereof.The properties of cisplatin, carboplatin, oxaliplatin, satraplatin,picoplatin, nedaplatin, triplatin tetranitrate, and lipoplatin are knownto those of ordinary skill in the art, and the active pharmaceuticalingredients and formulated products are commercially available. Wheate,et al., Dalton Trans. 2010, 39, 8113-27; Apps, et al., Endocrine-RelatedCancer 2015, 22, 219-233.

In an embodiment, the platinum-based antineoplastic drug is cisplatin,which has the chemical name (SP-4-2)-diamminedichloroplatinum(II)(Formula (12)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof. The preparation and properties of cisplatin aredescribed in, e.g., von Hoff and Rozencweig, Adv. Pharmacol. &Chemtherapy 1979, 16, 273-294.

In an embodiment, the platinum-based antineoplastic drug is carboplatin,which has the chemical namecis-diammine(cyclobutane-1,1-dicarboxylate-O,O′)platinum(II) (Formula(13)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof. The preparation and properties of carboplatin aredescribed in U.S. Pat. No. 4,140,707, the disclosure of which isincorporated by reference herein.

In an embodiment, the platinum-based antineoplastic drug is oxaliplatin,which has the chemical name[(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O′)platinum(II) orcis-[(1R,2R)-1,2-cyclohexanediamine-N,N] [oxalato(2-)-O₁O] platinum(Formula (14)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof. The preparation and properties of oxaliplatin aredescribed in U.S. Pat. Nos. 4,169,846; 5,420,319; and 5,716,988, thedisclosures of which are incorporated by reference herein.

In an embodiment, the platinum-based antineoplastic drug is satraplatin,which has the chemical name(OC-6-43)-bis(acetato)amminedichloro(cyclohexylamine)platinum orbis(acetato) ammine dichloro (cyclohexylamine) platinum(IV) (Formula(15)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof. The preparation and properties of satraplatin aredescribed in U.S. Pat. Nos. 5,072,011; 5,244,919; 6,518,428, thedisclosures of which are incorporated by reference herein.

In an embodiment, the platinum-based antineoplastic drug is picoplatin,which has the chemical name azane; 2-methylpyridine; platinum(2+);dichloride (Formula (16)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof. The preparation and properties of picoplatin aredescribed in U.S. Pat. Nos. 5,665,771 and 6,518,428; the disclosures ofwhich are incorporated by reference herein.

In an embodiment, the platinum-based antineoplastic drug is nedaplatin,which has the chemical name diammine[(hydroxy-κO)acetato(2−)-κO]platinum(Formula (17)):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof. Nedaplatin has been described in Alberts, et al.,Cancer Chemother. Pharmacol. 1997, 39, 493-497 and Wheate, et al.,Dalton Trans. 2010, 39, 8113-8127.

In an embodiment, the platinum-based antineoplastic drug is triplatin ora pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof. In an embodiment, the platinum drug is triplatintetranitrate (Formula (18)):

In an embodiment, the platinum-based antineoplastic drug is lipoplatin,which is a nanoparticle containing a combination of lipids andcisplatin. The clinical efficacy of lipoplatin is described inStathopoulos, et al., Cancer Chemtherapy & Pharmacol. 2011, 68, 945-950.The preparation, properties, and uses of lipoplatin are described inU.S. Pat. No. 6,511,676, the disclosure of which is incorporated byreference herein.

Pharmaceutical Compositions

In an embodiment, an active pharmaceutical ingredient or combination ofactive pharmaceutical ingredients, such as any of the foregoing EZH2inhibitors and optionally platinum-based antineoplastic drugs, isprovided as a pharmaceutically acceptable composition.

In some embodiments, the concentration of each of the activepharmaceutical ingredients provided in the pharmaceutical compositionsof the invention, such as any of the foregoing EZH2 inhibitors, is lessthan, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%,18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%,0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%,0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%,0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of thepharmaceutical composition.

In some embodiments, the concentration of each of the activepharmaceutical ingredients provided in the pharmaceutical compositionsof the invention, such as any of the foregoing EZH2 inhibitors, isgreater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%,19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%,16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%,14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%,11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%,9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%,6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%,3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%,0.3%, 0.2%, 01%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%,0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%,0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%,0.0002% or 0.0001% w/w, w/v, or v/v of the pharmaceutical composition.

In some embodiments, the concentration of each of the activepharmaceutical ingredients provided in the pharmaceutical compositionsof the invention, such as any of the foregoing EZH2 inhibitors, is inthe range from about 0.0001% to about 50%, about 0.001% to about 40%,about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% toabout 25%, about 0.07% to about 24%, about 0.08% to about 23%, about0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%,about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% toabout 14%, about 0.9% to about 12% or about 1% to about 10% w/w, w/v orv/v of the pharmaceutical composition.

In some embodiments, the concentration of each of the activepharmaceutical ingredients provided in the pharmaceutical compositionsof the invention, such as any of the foregoing EZH2 inhibitors, is inthe range from about 0.001% to about 10%, about 0.01% to about 5%, about0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%,about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% toabout 0.9% w/w, w/v or v/v of the pharmaceutical composition.

In some embodiments, the amount of each of the active pharmaceuticalingredients provided in the pharmaceutical compositions of theinvention, such as any of the foregoing EZH2 inhibitors, is equal to orless than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g,5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g,0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g,0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g,0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g,0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g,0.0002 g, or 0.0001 g.

In some embodiments, the amount of each of the active pharmaceuticalingredients provided in the pharmaceutical compositions of theinvention, such as any of the foregoing EZH2 inhibitors, is more than0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g,0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g,0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g,0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g,0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g,2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5g, 9 g, 9.5 g, or 10 g.

Each of the active pharmaceutical ingredients according to the inventionis effective over a wide dosage range. For example, in the treatment ofadult humans, dosages independently range from 0.01 to 1000 mg, from 0.5to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day areexamples of dosages that may be used. The exact dosage will depend uponthe route of administration, the form in which the compound isadministered, the gender and age of the subject to be treated, the bodyweight of the subject to be treated, and the preference and experienceof the attending physician. The clinically-established dosages of theforegoing EZH2 inhibitors may also be used if appropriate.

In an embodiment, the molar ratio of two active pharmaceuticalingredients in the pharmaceutical compositions is in the range from 10:1to 1:10, from 2.5:1 to 1:2.5, and about 1:1. In an embodiment, theweight ratio of the molar ratio of two active pharmaceutical ingredientsin the pharmaceutical compositions is selected from the group consistingof 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1,9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18,1:19, and 1:20. In an embodiment, the weight ratio of the molar ratio oftwo active pharmaceutical ingredients in the pharmaceutical compositionsis selected from the group consisting of 20:1, 19:1, 18:1, 17:1, 16:1,15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1,2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12,1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, and 1:20.

In an embodiment, the pharmaceutical compositions of the presentinvention, such as any of the foregoing EZH2 inhibitors, are for use inthe treatment of cancers associated with overexpression or amplificationof CARM1. In an embodiment, the pharmaceutical compositions of thepresent invention are for use in the treatment of a cancer associatedwith overexpression or amplification of CARM1 selected from the groupconsisting of bladder cancer, squamous cell carcinoma including head andneck cancer, pancreatic ductal adenocarcinoma, pancreatic cancer, coloncarcinoma, mammary carcinoma, breast cancer, fibrosarcoma, mesothelioma,renal cell carcinoma, lung carcinoma, thyoma, prostate cancer,colorectal cancer, ovarian cancer, acute myeloid leukemia, thymuscancer, brain cancer, squamous cell cancer, skin cancer, eye cancer,retinoblastoma, melanoma, intraocular melanoma, oral cavity andoropharyngeal cancers, gastric cancer, stomach cancer, cervical cancer,renal cancer, kidney cancer, liver cancer, esophageal cancer, testicularcancer, gynecological cancer, thyroid cancer, acquired immune deficiencysyndrome (AIDS)-related lymphoma, Kaposi's sarcoma, viral-inducedcancer, glioblastoma, esophageal tumors, hematological neoplasms,non-small-cell lung cancer, chronic myelocytic leukemia, diffuse largeB-cell lymphoma, esophagus tumor, follicle center lymphoma, head andneck tumor, hepatitis C virus infection, hepatocellular carcinoma,Hodgkin's disease, metastatic colon cancer, multiple myeloma,non-Hodgkin's lymphoma, indolent non-Hodgkin's lymphoma, ovary tumor,pancreas tumor, renal cell carcinoma, small-cell lung cancer, stage IVmelanoma, chronic lymphocytic leukemia, B-cell acute lymphoblasticleukemia (ALL), mature B-cell ALL, follicular lymphoma, mantle celllymphoma, and Burkitt's lymphoma.

Described below are non-limiting pharmaceutical compositions and methodsfor preparing the same.

Pharmaceutical Compositions for Oral Administration

In some embodiments, the invention provides a pharmaceutical compositionfor oral administration containing the active pharmaceutical ingredientor combination of active pharmaceutical ingredients, such as the EZH2inhibitors described herein, and a pharmaceutical excipient suitable fororal administration.

In some embodiments, the invention provides a solid pharmaceuticalcomposition for oral administration containing: (i) an effective amountof an active pharmaceutical ingredient or combination of activepharmaceutical ingredients, and (ii) a pharmaceutical excipient suitablefor oral administration. In selected embodiments, the compositionfurther contains (iii) an effective amount of a third activepharmaceutical ingredient and optionally (iv) an effective amount of afourth active pharmaceutical ingredient.

In some embodiments, the pharmaceutical composition may be a liquidpharmaceutical composition suitable for oral consumption. Pharmaceuticalcompositions of the invention suitable for oral administration can bepresented as discrete dosage forms, such as capsules, sachets, ortablets, or liquids or aerosol sprays each containing a predeterminedamount of an active ingredient as a powder or in granules, a solution,or a suspension in an aqueous or non-aqueous liquid, an oil-in-wateremulsion, a water-in-oil liquid emulsion, powders for reconstitution,powders for oral consumptions, bottles (including powders or liquids ina bottle), orally dissolving films, lozenges, pastes, tubes, gums, andpacks. Such dosage forms can be prepared by any of the methods ofpharmacy, but all methods include the step of bringing the activeingredient(s) into association with the carrier, which constitutes oneor more necessary ingredients. In general, the compositions are preparedby uniformly and intimately admixing the active ingredient(s) withliquid carriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired presentation. Forexample, a tablet can be prepared by compression or molding, optionallywith one or more accessory ingredients. Compressed tablets can beprepared by compressing in a suitable machine the active ingredient in afree-flowing form such as powder or granules, optionally mixed with anexcipient such as, but not limited to, a binder, a lubricant, an inertdiluent, and/or a surface active or dispersing agent. Molded tablets canbe made by molding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent.

Pharmaceutical Compositions for Injection

In some embodiments, the invention provides a pharmaceutical compositionfor injection containing an active pharmaceutical ingredient orcombination of active pharmaceutical ingredients, such as an EZH2inhibitor and optionally a platinum-based antineoplastic drug, and apharmaceutical excipient suitable for injection.

The forms in which the compositions of the present invention may beincorporated for administration by injection include aqueous or oilsuspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, orpeanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueoussolution, and similar pharmaceutical vehicles.

Aqueous solutions in saline are also conventionally used for injection.Ethanol, glycerol, propylene glycol and liquid polyethylene glycol (andsuitable mixtures thereof), cyclodextrin derivatives, and vegetable oilsmay also be employed. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, for the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid and thimerosal.

Sterile injectable solutions are prepared by incorporating an activepharmaceutical ingredient or combination of active pharmaceuticalingredients in the required amounts in the appropriate solvent withvarious other ingredients as enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, certaindesirable methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Pharmaceutical Compositions for Topical Delivery

In some embodiments, the invention provides a pharmaceutical compositionfor transdermal delivery containing an active pharmaceutical ingredientor combination of active pharmaceutical ingredients, such as the EZH2inhibitors described herein, and a pharmaceutical excipient suitable fortransdermal delivery.

Compositions of the present invention can be formulated intopreparations in solid, semi-solid, or liquid forms suitable for local ortopical administration, such as gels, water soluble jellies, creams,lotions, suspensions, foams, powders, slurries, ointments, solutions,oils, pastes, suppositories, sprays, emulsions, saline solutions,dimethylsulfoxide (DMSO)-based solutions. In general, carriers withhigher densities are capable of providing an area with a prolongedexposure to the active ingredients. In contrast, a solution formulationmay provide more immediate exposure of the active ingredient to thechosen area.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients, which are compounds that allow increasedpenetration of, or assist in the delivery of, therapeutic moleculesacross the stratum corneum permeability barrier of the skin. There aremany of these penetration-enhancing molecules known to those trained inthe art of topical formulation. Examples of such carriers and excipientsinclude, but are not limited to, humectants (e.g., urea), glycols (e.g.,propylene glycol), alcohols (e.g., ethanol), fatty acids (e.g., oleicacid), surfactants (e.g., isopropyl myristate and sodium laurylsulfate), pyrrolidones, glycerol monolaurate, sulfoxides, terpenes(e.g., menthol), amines, amides, alkanes, alkanols, water, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin, and polymers such as polyethylene glycols.

Another exemplary formulation for use in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of an active pharmaceutical ingredient or combination of activepharmaceutical ingredients in controlled amounts, either with or withoutanother active pharmaceutical ingredient.

The construction and use of transdermal patches for the delivery ofpharmaceutical agents is well known in the art. See, e.g., U.S. Pat.Nos. 5,023,252; 4,992,445 and 5,001,139. Such patches may be constructedfor continuous, pulsatile, or on demand delivery of pharmaceuticalagents.

Pharmaceutical Compositions for Inhalation

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra and the EZH2 inhibitors described herein. The compositions areadministered by the oral or nasal respiratory route for local orsystemic effect. Compositions in pharmaceutically acceptable solventsmay be nebulized by use of inert gases. Nebulized solutions may beinhaled directly from the nebulizing device or the nebulizing device maybe attached to a face mask tent, or intermittent positive pressurebreathing machine. Solution, suspension, or powder compositions may beadministered, orally or nasally, from devices that deliver theformulation in an appropriate manner. Dry powder inhalers may also beused to provide inhaled delivery of the compositions.

Other Pharmaceutical Compositions

Pharmaceutical compositions of the EZH2 inhibitors described herein mayalso be prepared from compositions described herein and one or morepharmaceutically acceptable excipients suitable for sublingual, buccal,rectal, intraosseous, intraocular, intranasal, epidural, or intraspinaladministration. Preparations for such pharmaceutical compositions arewell-known in the art. See, e.g., Anderson, et al., eds., Handbook ofClinical Drug Data, Tenth Edition, McGraw-Hill, 2002; and Pratt andTaylor, eds., Principles of Drug Action, Third Edition, ChurchillLivingston, 1990, each of which is incorporated by reference herein inits entirety.

Administration of an active pharmaceutical ingredient or combination ofactive pharmaceutical ingredients or a pharmaceutical compositionthereof can be effected by any method that enables delivery of thecompounds to the site of action. These methods include oral routes,intraduodenal routes, parenteral injection (including intravenous,intraarterial, subcutaneous, intramuscular, intravascular,intraperitoneal or infusion), topical (e.g., transdermal application),via local delivery by catheter or stent or through inhalation. Theactive pharmaceutical ingredient or combination of active pharmaceuticalingredients can also be administered intrathecally.

The compositions of the invention may also be delivered via animpregnated or coated device such as a stent, for example, or anartery-inserted cylindrical polymer. Such a method of administrationmay, for example, aid in the prevention or amelioration of restenosisfollowing procedures such as balloon angioplasty. Without being bound bytheory, compounds of the invention may slow or inhibit the migration andproliferation of smooth muscle cells in the arterial wall whichcontribute to restenosis. A compound of the invention may beadministered, for example, by local delivery from the struts of a stent,from a stent graft, from grafts, or from the cover or sheath of a stent.In some embodiments, a compound of the invention is admixed with amatrix. Such a matrix may be a polymeric matrix, and may serve to bondthe compound to the stent. Polymeric matrices suitable for such use,include, for example, lactone-based polyesters or copolyesters such aspolylactide, polycaprolactonglycolide, polyorthoesters, polyanhydrides,polyaminoacids, polysaccharides, polyphosphazenes, poly(ether-ester)copolymers (e.g., PEO-PLLA); polydimethylsiloxane,poly(ethylene-vinylacetate), acrylate-based polymers or copolymers(e.g., polyhydroxyethyl methylmethacrylate, polyvinyl pyrrolidinone),fluorinated polymers such as polytetrafluoroethylene and celluloseesters. Suitable matrices may be nondegrading or may degrade with time,releasing the compound or compounds. The active pharmaceuticalingredient or combination of active pharmaceutical ingredients may beapplied to the surface of the stent by various methods such as dip/spincoating, spray coating, dip-coating, and/or brush-coating. The compoundsmay be applied in a solvent and the solvent may be allowed to evaporate,thus forming a layer of compound onto the stent. Alternatively, thecompound may be located in the body of the stent or graft, for examplein microchannels or micropores. When implanted, the compound diffusesout of the body of the stent to contact the arterial wall. Such stentsmay be prepared by dipping a stent manufactured to contain suchmicropores or microchannels into a solution of the compound of theinvention in a suitable solvent, followed by evaporation of the solvent.Excess drug on the surface of the stent may be removed via an additionalbrief solvent wash. In yet other embodiments, compounds of the inventionmay be covalently linked to a stent or graft. A covalent linker may beused which degrades in vivo, leading to the release of the compound ofthe invention. Any bio-labile linkage may be used for such a purpose,such as ester, amide or anhydride linkages. The active pharmaceuticalingredient or combination of active pharmaceutical ingredients mayadditionally be administered intravascularly from a balloon used duringangioplasty. Extravascular administration of an active pharmaceuticalingredient or combination of active pharmaceutical ingredients via thepericard or via advential application of formulations of the inventionmay also be performed to decrease restenosis.

Exemplary parenteral administration forms include solutions orsuspensions of active compound in sterile aqueous solutions, forexample, aqueous propylene glycol or dextrose solutions. Such dosageforms can be suitably buffered, if desired.

The invention also provides kits. The kits include an activepharmaceutical ingredient or combination of active pharmaceuticalingredients, either alone or in combination in suitable packaging, andwritten material that can include instructions for use, discussion ofclinical studies and listing of side effects. Such kits may also includeinformation, such as scientific literature references, package insertmaterials, clinical trial results, and/or summaries of these and thelike, which indicate or establish the activities and/or advantages ofthe composition, and/or which describe dosing, administration, sideeffects, drug interactions, or other information useful to the healthcare provider. Such information may be based on the results of variousstudies, for example, studies using experimental animals involving invivo models and studies based on human clinical trials. The kit mayfurther contain another active pharmaceutical ingredient. In selectedembodiments, an active pharmaceutical ingredient or combination ofactive pharmaceutical ingredients are provided as separate compositionsin separate containers within the kit. In selected embodiments, anactive pharmaceutical ingredient or combination of active pharmaceuticalingredients are provided as a single composition within a container inthe kit. Suitable packaging and additional articles for use (e.g.,measuring cup for liquid preparations, foil wrapping to minimizeexposure to air, and the like) are known in the art and may be includedin the kit. Kits described herein can be provided, marketed and/orpromoted to health providers, including physicians, nurses, pharmacists,formulary officials, and the like. Kits may also, in selectedembodiments, be marketed directly to the consumer.

In some embodiments, the invention provides a kit comprising acomposition comprising a therapeutically effective amount of an activepharmaceutical ingredient or combination of active pharmaceuticalingredients or a pharmaceutically acceptable salt, solvate, hydrate,cocrystal, or prodrug thereof. These compositions are typicallypharmaceutical compositions. The kit is for co-administration of theactive pharmaceutical ingredient or combination of active pharmaceuticalingredients, either simultaneously or separately.

In some embodiments, the invention provides a kit comprising (1) acomposition comprising a therapeutically effective amount of an activepharmaceutical ingredient or combination of active pharmaceuticalingredients or a pharmaceutically acceptable salt, solvate, hydrate,cocrystal, or prodrug thereof, and (2) a diagnostic test for determiningwhether a patient's cancer is a particular subtype of a cancer. Any ofthe foregoing diagnostic methods may be utilized in the kit.

The kits described above are for use in the treatment of the diseasesand conditions described herein. In an embodiment, the kits are for usein the treatment of cancer. In some embodiments, the kits are for use intreating solid tumor cancers.

In an embodiment, the kits of the present invention are for use in thetreatment of cancer. In an embodiment, the kits of the present inventionare for use in the treatment of a cancer selected from the groupconsisting of bladder cancer, squamous cell carcinoma including head andneck cancer, pancreatic ductal adenocarcinoma (PDA), pancreatic cancer,colon carcinoma, mammary carcinoma, breast cancer, fibrosarcoma,mesothelioma, renal cell carcinoma, lung carcinoma, thyoma, prostatecancer, colorectal cancer, ovarian cancer, acute myeloid leukemia,thymus cancer, brain cancer, squamous cell cancer, skin cancer, eyecancer, retinoblastoma, melanoma, intraocular melanoma, oral cavity andoropharyngeal cancers, gastric cancer, stomach cancer, cervical cancer,renal cancer, kidney cancer, liver cancer, ovarian cancer, esophagealcancer, testicular cancer, gynecological cancer, thyroid cancer, aquiredimmune deficiency syndrome (AIDS)-related cancers (e.g., lymphoma andKaposi's sarcoma), viral-induced cancer, glioblastoma, esophagealtumors, hematological neoplasms, non-small-cell lung cancer, chronicmyelocytic leukemia, diffuse large B-cell lymphoma, esophagus tumor,follicle center lymphoma, head and neck tumor, hepatitis C virusinfection, hepatocellular carcinoma, Hodgkin's disease, metastatic coloncancer, multiple myeloma, non-Hodgkin's lymphoma, indolent non-Hodgkin'slymphoma, ovary tumor, pancreas tumor, renal cell carcinoma, small-celllung cancer, stage IV melanoma, chronic lymphocytic leukemia, B-cellacute lymphoblastic leukemia (ALL), mature B-cell ALL, follicularlymphoma, mantle cell lymphoma, and Burkitt's lymphoma.

Dosages and Dosing Regimens

The amounts of the pharmaceutical compositions administered using themethods herein, such as the dosages of EZH2 inhibitors, will bedependent on the human or mammal being treated, the severity of thedisorder or condition, the rate of administration, the disposition ofthe active pharmaceutical ingredients and the discretion of theprescribing physician. However, an effective dosage is in the range ofabout 0.001 to about 100 mg per kg body weight per day, such as about 1to about 35 mg/kg/day, in single or divided doses. For a 70 kg human,this would amount to about 0.05 to 7 g/day, such as about 0.05 to about2.5 g/day. In some instances, dosage levels below the lower limit of theaforesaid range may be more than adequate, while in other cases stilllarger doses may be employed without causing any harmful sideeffect—e.g., by dividing such larger doses into several small doses foradministration throughout the day. The dosage of the pharmaceuticalcompositions and active pharmaceutical ingredients may be provided inunits of mg/kg of body mass or in mg/m² of body surface area.

In some embodiments, the invention includes a methods of treating acancer in a human subject suffering from the cancer in which cancercells overexpress arginine methyltransferase CARM1, the methodcomprising the steps of administering a therapeutically effective doseof an active pharmaceutical ingredient that is an EZH2 inhibitor and anactive pharmaceutical ingredient that is an platinum-basedantineoplastic drug to the human subject. In some embodiments, the EZH2inhibitor is administered before the platinum-based antineoplastic drug.In some embodiments, the platinum-based antineoplastic drug isadministered concurrently with the EZH2 inhibitor. In some embodiments,the EZH2 inhibitor is administered after the platinum-basedantineoplastic drug.

In some embodiments, a pharmaceutical composition or activepharmaceutical ingredient is administered in a single dose. Suchadministration may be by injection, e.g., intravenous injection, inorder to introduce the active pharmaceutical ingredient quickly.However, other routes, including the oral route, may be used asappropriate. A single dose of a pharmaceutical composition may also beused for treatment of an acute condition.

In some embodiments, a pharmaceutical composition or activepharmaceutical ingredient is administered in multiple doses. In anembodiment, a pharmaceutical composition is administered in multipledoses. Dosing may be once, twice, three times, four times, five times,six times, or more than six times per day. Dosing may be once a month,once every two weeks, once a week, or once every other day. In otherembodiments, a pharmaceutical composition is administered about once perday to about 6 times per day. In some embodiments, a pharmaceuticalcomposition is administered once daily, while in other embodiments, apharmaceutical composition is administered twice daily, and in otherembodiments a pharmaceutical composition is administered three timesdaily.

Administration of the active pharmaceutical ingredients in the methodsof the invention may continue as long as necessary. In selectedembodiments, a pharmaceutical composition is administered for more than1, 2, 3, 4, 5, 6, 7, 14, or 28 days. In some embodiments, apharmaceutical composition is administered for less than 28, 14, 7, 6,5, 4, 3, 2, or 1 day. In some embodiments, a pharmaceutical compositionis administered chronically on an ongoing basis—e.g., for the treatmentof chronic effects. In some embodiments, the administration of apharmaceutical composition continues for less than about 7 days. In yetanother embodiment the administration continues for more than about 6,10, 14, 28 days, two months, six months, or one year. In some cases,continuous dosing is achieved and maintained as long as necessary.

In some embodiments, an effective dosage of an active pharmaceuticalingredient disclosed herein is in the range of about 1 mg to about 500mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25mg to about 200 mg, about 10 mg to about 200 mg, about 20 mg to about150 mg, about 30 mg to about 120 mg, about 10 mg to about 90 mg, about20 mg to about 80 mg, about 30 mg to about 70 mg, about 40 mg to about60 mg, about 45 mg to about 55 mg, about 48 mg to about 52 mg, about 50mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, about95 mg to about 105 mg, about 150 mg to about 250 mg, about 160 mg toabout 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about198 to about 202 mg. In some embodiments, an effective dosage of anactive pharmaceutical ingredient disclosed herein is about 25 mg, about50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175mg, about 200 mg, about 225 mg, or about 250 mg.

In some embodiments, an effective dosage of an active pharmaceuticalingredient disclosed herein is in the range of about 0.01 mg/kg to about4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about3.2 mg/kg, about 0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg toabout 2.85 mg/kg, about 0.3 mg to about 2.15 mg/kg, about 0.45 mg/kg toabout 1.7 mg/kg, about 0.15 mg/kg to about 1.3 mg/kg, about 0.3 mg/kg toabout 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg toabout 0.85 mg/kg, about 0.65 mg/kg to about 0.8 mg/kg, about 0.7 mg/kgto about 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15mg/kg to about 1.7 mg/kg, about 1.3 mg/kg mg to about 1.6 mg/kg, about1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg,about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg,about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg,about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95mg/kg. In some embodiments, an effective dosage of an activepharmaceutical ingredient disclosed herein is about 0.35 mg/kg, about0.7 mg/kg, about 1 mg/kg, about 1.4 mg/kg, about 1.8 mg/kg, about 2.1mg/kg, about 2.5 mg/kg, about 2.85 mg/kg, about 3.2 mg/kg, or about 3.6mg/kg.

In some embodiments, an effective dosage of an active pharmaceuticalingredient disclosed herein is in the range of about 1 mg to about 500mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mgto about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg,about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg toabout 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, orabout 198 to about 207 mg. In some embodiments, an effective dosage ofan active pharmaceutical ingredient disclosed herein is about 25 mg,about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg,about 175 mg, about 200 mg, about 225 mg, or about 250 mg.

In some embodiments, an active pharmaceutical ingredient is administeredat a dosage of 10 to 200 mg BID, including 50, 60, 70, 80, 90, 100, 150,or 200 mg BID. In some embodiments, an active pharmaceutical ingredientis administered at a dosage of 10 to 500 mg BID, including 1, 5, 10, 15,25, 50, 75, 100, 150, 200, 300, 400, or 500 mg BID.

In some instances, dosage levels below the lower limit of the aforesaidranges may be more than adequate, while in other cases still largerdoses may be employed without causing any harmful side effect—e.g., bydividing such larger doses into several small doses for administrationthroughout the day.

An effective amount of the combination of the active pharmaceuticalingredient may be administered in either single or multiple doses by anyof the accepted modes of administration of agents having similarutilities, including rectal, buccal, intranasal and transdermal routes,by intra-arterial injection, intravenously, intraperitoneally,parenterally, intramuscularly, subcutaneously, orally, topically, or asan inhalant.

EXAMPLES

The embodiments encompassed herein are now described with reference tothe following examples. These examples are provided for the purpose ofillustration only and the disclosure encompassed herein should in no waybe construed as being limited to these examples, but rather should beconstrued to encompass any and all variations which become evident as aresult of the teachings provided herein.

Example 1—CARM1 is Associated with Poor Prognosis in Ovarian CancerPatients

In FIG. 1, CARM1 expression in different types of cancer is illustrated.Significant amplification is observed in a number of cancers, includingovarian, uterine, breast, sarcoma, and pancreatic cancers and glioma. InFIG. 2, overall survival in CARM1-expressing cells is shown to be poor.Data was obtained from the Cancer Genome Atlas. FIG. 3 illustrates thelack of co-occurrence of CARM1 amplification with mutations in thehomologous recombination (HR) DNA repair genes BRCA1 and BRCA2. Overall,the results indicate that CARM1 is amplified in a variety of cancers,including 10% of high-grade ovarian carcinomas. CARM1 amplification doesnot co-occur with mutations in the HR DNA repair pathway, and CARM1amplification is associated with poor prognosis.

Example 2—CARM1-Expressing Cells are Sensitive to the EZH2 InhibitorGSK126

In FIG. 4, CARM1 expression in a panel of high-grade ovarian carcinomasis depicted. FIG. 5 illustrates the results of a colony formation assaywith a panel of normal and ovarian cancer cell lines with DMSO and with10 μM of the EZH2 inhibitor GSK126 (Formula (1)). The data in FIG. 5 isquantitatively assessed in FIG. 6, with both figures illustrating theparticular sensitivity of CARM1-expressing cells to the EZH2 inhibitor.

In FIG. 7, response curves for GSK126 in a panel of normal and ovariancancer cell lines are shown. The IC₅₀ values determined from thesecurves for the panel of normal and ovarian cancer cell lines are listedin FIG. 8. Significantly reduced IC₅₀ values are observed for theCARM1-expressing cell lines (A1847, OVCAR10, and PEO4).

Overall, the results demonstrate the surprising finding thatCARM1-expressing cells are sensitive to the EZH2 inhibitor GSK126.Sensitization of CARM1-expressing cells to an EZH2 inhibitor may befurther exploited by administration of one or more platinum-basedantineoplastic drugs.

Example 3—CARM1 Depletion Abrogates GSK126 Sensitivity inCARM1-Expressing Cells

To confirm the relationship between CARM1 overexpression and sensitivityto EZH2 inhibition, further studies were performed to assess the impactof CARM1 depletion. In FIG. 9, a Western blot of CARM1 and BAF155me2 inCARM1 depleted cells with a control (Actin) is shown. In addition, theresults of a colony formation assay (unstained, magnified) with normaland CARM1-depleted cells, in DMSO (control) and treated with GSK126, areshown in FIG. 10. A colony formation assay with normal andCARM1-depleted cells, in DMSO (control) and after treatment with GSK126is shown in FIG. 11, with the results quantified in FIG. 12. Finally,FIG. 13 illustrates the results of treatment of CARM1 depleted cellswith GSK126. Together, the results indicate that CARM1 depletion leadsto decreased BAF155 methylation, while also abrogating the sensitivityof the EZH2 inhibitor GSK126 in CARM1-expressing cells.

Example 4—CARM1-Expressing Ovarian Cancer Depends on the HistoneMethyltransferase EZH2 Activity

In this example, it is shown that EZH2 inhibition is selective againstCARM1 expression in epithelial ovarian cancer. High CARM1 expressionpredicts a shorter survival in ovarian cancer patients. Inhibition ofEZH2 activity using a clinically applicable small molecule inhibitorsignificantly suppressed the growth of CARM1-expressing, but not CARM1deficient, ovarian tumors in two xenograft models and improved thesurvival of mice bearing CARM1-expressing tumors. The observedselectivity correlates with upregulation of EZH2 target genes in aCARM1-dependent manner. CARM1 promotes EZH2 dependent gene silencing ofEZH2/BAF155 target tumor suppressor genes by methylating BAF155 to alterthe antagonism between EZH2 and BAF155. Together, these results indicatethat pharmacological inhibition of EZH2 is a novel therapeutic strategyfor CARM1-expressing cancers.

Methods

Cell lines and culture conditions. Human EOC cell lines were obtainedfrom ATCC within 3 years or obtained as described in Li, et al. MCR(2010) 8:1610-8, and were re-authenticated by The Wistar Institute'sGenomics Facility at the end of experiments within last three monthsusing short tandem repeat profiling using AmpFISTR Identifiler PCRAmplification kit (Life Technologies) and cultured as described in Li,et al. MCR (2010) 8:1610-8. Human ovarian surface epithelial cells wereobtained and cultured as described in Li, et al. MCR (2010) 8:1610-8,Mycoplasma testing was performed by LookOut Mycoplasma PCR detection(Sigma).

Reagents and Antibodies. Small molecules used in the epigenetic screenwere obtained from Structural Genomics Consortium or The WistarInstitute Molecular Screening Facility. GSK126 was obtained from ActiveBiochem or Xcess Biosciences. Antibodies were obtained from: mouseanti-CARM1 (Cell Signaling, Cat. No: 12495, 1:1000 for immunoblotting),goat anti-BAF155 (Santa Cruz, Cat. No: SC9746, 1:1000 forimmunoblotting), rabbit anti-methylated R1064 BAF155 (Millipore, Cat.No: ABE1339, 1:1000 for immunoblotting), rabbit anti-EZH2 (CellSignaling, Cat. No: 5246, 1:1000 for immunoblotting), rabbitanti-cleaved PARP p85 (Promega, Cat. No: G7341, 1:1000 forimmunoblotting), mouse anti-Ki67 (Cell Signaling, Cat. No: 9449, 1:500for IHC), rabbit anti-cleaved caspase 3 (Cell Signaling, Cat. No: 9661,1:1000 for immunoblotting and 1:50 for IHC), rabbit anti-H3K27Me3 (CellSignaling, Cat. No: 9733, 1:1000 for immunoblotting and 1:100 for IHC),mouse anti-β-actin (Sigma, Cat. No: A1978, 1:20,000 for immunoblotting),rabbit anti-RNA pol II (Santa Cruz, Cat. No: sc-899). Growth factorreduced basement membrane matrix (Matrigel) was obtained from Corning.

CRISPR-mediated CARM1 knockout. pLentiCRISPR-CARM1 was constructed byinserting the CARM1 guide RNA (gRNA; 5′-AGCACGGAAAATCTACGCGG-3′ (SEQ IDNO: 1)) according to Shalem, et al. Science (2014) 343:84-7. In brief,pLentiCRISPR v2 (Addgene) was digested and dephosphorylated with BsmBIrestriction enzyme (Fermentas) for 30 min at 37° C. The digested plasmidwas run on a 1% agarose gel, cut out, and purified using the Wizard SVGel and PCR Clean Up kit (Promega). The oligonucleotides werephosphorylated using T4 PNK (M0201S) with T4 Ligation Buffer (NewEngland Biolabs, Inc.). Samples were annealed in a thermocycler at 37°C. for 30 min and then at 95° C. for 5 min and then were ramped down to25° C. at 5° C./min. Annealed oligonucleotides were diluted 1:200 inRNase/DNase-free water. Ligation of the annealed oligonucleotide anddigested pLentiCRISPR v2 plasmid was performed using Quick Ligase (NewEngland Biolabs, Inc.).

Lentivirus and retrovirus infection. Retrovirus production andtransduction were performed as described in Bitler et al. NatureMedicine (2015) 21:231-8 and Aird et al. Cell Reports (2013) 3:1252-65.Phoenix cells were used to package the viruses. Lentivirus was packagedusing the Virapower Kit from Invitrogen according to the manufacturer'sinstructions as described in Li, et al. MCR (2010) 8:1610-8 and Ye, etal. Mol. Cell Biol. (2007) 27:2452-65. The following shRNAs obtainedfrom the Molecular Screening Facility at The Wistar Institute were used:pLKO.1-shCARM1 (TRCN0000059090 and TRCN0000059090), pLKO.1-shEZH2(TRCN0000040073) and pLKO.1-shBAF155 (TRCN00001353636). Cells infectedwith viruses encoding the puromycin resistance gene were selected in 1μg/ml puromycin.

Reverse-transcriptase quantitative PCR (RT-qPCR). RNA was isolated byRNeasy Mini Kit (Qiagen). mRNA relative expression for DAB2, DLC1, andPMAIP was determined using SYBR green 1-step iScript (Bio-Rad) with aLife Technologies QuantStudio 3. The primers were:5′-TTCATTGCCCGTGATGTGACA-3′ (DAB2 forward (SEQ ID NO: 2)) and5′-CCTGTTGCCCGGTTTTTATGG-3′ (DAB2 reverse (SEQ ID NO: 3));5′-AACCCAAGACTACGGCTATTCA-3′ (DLC1 forward (SEQ ID NO: 4)) and5′-CATAAAGCTGTGCATACTGGGG-3′ (DLC1 reverse (SEQ ID NO: 5));5′-ACCAAGCCGGATTTGCGATT-3′ (NOXA forward (SEQ ID NO: 6)) and5′-ACTTGCACTTGTTCCTCGTGG-3′ (NOXA reverse (SEQ ID NO: 7)); and5′-CATGTGCAGTACATCCATACGG-3′ (TIMP3 forward (SEQ ID NO: 8)) and5′-CATCATAGACGCGACCTGTCA-3′ (TIMP3 reverse (SEQ ID NO: 9)).

Annexin V Assay. Phosphatidylserine externalization was detected usingan Annexin V staining kit (Millipore) following the manufacturer'sinstructions. Annexin V-positive cells were detected using the GuavaSystem and analyzed with the Guava Nexin software Module (Millipore).

3D Matrigel Assays. Matrigel was coated on the bottom of 8-well chamberslides and cells were plated on the matrigel (4,000 cells/well) in a 3%Matrigel/Media mixture. Media, matrigel and treatment (drug/vehicle)were replenished every fourth day. On day 12, five bright-field imageswere captured from each well/treatment. Acini diameter was measured fromimages with Image-J software (NTH). Also, on day 12 cell recoverysolution (BD) was used to remove acini from Matrigel, treated withtrypsin and total number of cells was counted for each treatment.

Colony formation assay. 500 to 5,000 cell were plated into a 24 welltissue culture plate and treated with the indicated compounds. Mediumwas changed every three days with appropriate drug doses for 12 days oruntil control wells became confluent. Colonies were washed twice withPBS and fixed with 10% methanol and 10% acetic acid in distilled water.Fixed colonies were stained with 0.005% crystal violet. Integrateddensity was measured using NIH ImageJ software.

Intrabursal orthotopic xenograft models in vivo. The protocols wereapproved by the Institutional Animal Care and Use Committee (IACUC). Forin vivo experiments, the sample size of 5 mice per group was determinedbased on the data shown from in vitro experiments. Intrabursalorthotopic xenograft was performed as described in Bitler, et al. NatureMedicine (2015) 21:231-8 and Bitler et al. Cancer Research (2011)71:6184-94. Briefly, 1×10⁶ A1847 parental or A1847 CARM1 knockout cellswere unilaterally injected into the ovarian bursa of 6-8-week old femaleimmunocompromised NSG mice (n=5 per group). One week after injection themice were randomized into two groups and treated with vehicle control(20% captisol) or GSK126 (50 mg/kg daily) for three weeks. At the end ofthe experiments, tumors were surgically dissected and tumor burden wascalculated based on tumor weight. For survival experiments, afterstopping the treatment, the mice were followed for mortality or when thetumor burden reached 10% of body weight as determined by The WistarInstitute IACUC guideline.

Subcutaneous xenograft models in vivo. The protocols were approved bythe Institutional Animal Care and Use Committee (IACUC). 5×10⁶ controlA1847 or A1847 CARM1 knockout cells were unilaterally injectedsubcutaneously into 6-8-week old female immunocompromised NSG mice (n=5per group). One week after injection the mice were randomized andtreated with vehicle control (20% captisol) or GSK126 (50 mg/kg daily).Tumor size was measured every 3 days for 3 weeks. At end of theexperiments, tumors were surgically dissected and tumor burden wascalculated based on tumor weight.

Epigenetic targeting small molecule set screen. A1847 parental and CARM1knockout cells were plated in 24-well plates and treated with 24epigenetic compounds. Cell medium was changed every three days withappropriate drug doses for 14 days or until control wells becameconfluent. Colonies were washed twice with PBS and fixed with 10%methanol and 10% acetic acid in distilled water. Fixed colonies werestained with 0.005% crystal violet. Integrated density was measuredusing NIH ImageJ software as a surrogate for cell growth.

RNA sequencing (RNA-seq) and chromatin immunoprecipitation followed bysequencing (ChIP-seq). RNA was extracted with Trizol (Invitrogen) andsubsequently cleaned and DNase-treated using RNeasy columns (Qiagen).DNase treated RNA was subjected to library preparation. Libraries forRNA-seq were prepared with ScriptSeq complete Gold kit (Epicentre) andsubjected to a 75 bp paired-end sequencing run on NextSeq 500, usingIllumina's NextSeq 500 high output sequencing kit following themanufacturer's instructions.

For ChIP-seq, cells were cross-linked with 1% formaldehyde for 10 min,followed by quenching with 125 mM glycine for 5 min. Fixed cells wereresuspended in cell lysis buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl,0.5% NP-40) and incubated on ice for 10 min. The lysates were washedwith MNase digestion buffer (20 mM Tris-HCl, pH7.5, 15 mM NaCl, 60 mMKCl, 1 mM CaCl₂) once and incubated for 20 minutes at 37° C. in thepresence of 1,000 Gel units of MNase (NEB, M0247S) in 250 μL reactionvolume. After adding the same volume of sonication buffer (100 mMTris-HCl, pH8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% sodiumdeoxycholate), the lysates were sonicated for 5 min (30 sec-on/30sec-off) in a Diagenode bioruptor and centrifuged at 15,000 rpm for 10min. The cleared supernatant equivalent to 2-4×10⁶ cells was incubatedwith 2.5 μg of anti-EZH2 antibody (Cell Signaling, Cat. No. 5246) or 2μg anti-H3K27Me3 antibody (Cell Signaling, Cat. No. 9733) on a rockerovernight. Bound chromatin was eluted and reverse-crosslinked at 65° C.overnight. For next-generation sequencing, ChIP-seq libraries wereprepared from 10 ng of ChIP and input DNA with the Ovation Ultralow DRMultiplex system (NuGEN). The ChIP-seq libraries were sequenced in a 51base pairs paired end run using the Illumina HiSeq 2000.

Chromatin Immunoprecipitation (ChIP). ChIP was performed as described inTu et al. Developmental Cell (2011) 21:1077-91. The following antibodieswere used to perform ChIP: anti-H3K27Me3 (Cell Signaling, Cat. No:9733), anti-BAF155 (Santa Cruz, Cat. No: sc-9746), anti-RNA polymeraseII (Santa Cruz, Cat. No: sc-899) or anti-EZH2 (Cell Signaling, Cat. No:5246). An isotype matched IgG was used as a negative control. ChIP DNAwas analyzed by quantitative PCR against the promoter of the indicatedgenes using the following primers: DAB2 Forward:5′-GTGTTCGGGGAGAAGTCAAA-3′ (SEQ ID NO: 10) and DAB2 Reverse:5′-ACGGATCTGTGAAACGAAGC-3′ (SEQ ID NO: 11); DLC1 Forward:5′-AAAATTTCCAAGCGCCACTA-3′ (SEQ ID NO: 12) and DLC1 Reverse:5′-ACACCGCCTTCTACCTTCCT-3′ (SEQ ID NO: 13); NOXA Forward:5′-TATTGTGGGAGGTGGGGATA-3′ (SEQ ID NO: 14) and NOXA Reverse:5′-GGCCTGAAAACTTACGATGG-3′ (SEQ ID NO: 15); TIMP3 Forward:5′-ACTCCCCTACGCAAGGATTC-3′ (SEQ ID NO: 16) and TIMP3 Reverse:5′-CGTGTGAAGGCAGTTTGGTT-3′ (SEQ ID NO: 17).

Bioinformatics Analysis. RNA-seq data was aligned using bowtie2 (see,e.g., Langmead, et al. Nat Methods (2012) 9:357-9) against hg19 versionof the human genome and RSEM v1.2.12 software (see, e.g., Li et al. BMCBioinformatics (2011) 12:323) was used to estimate raw read counts andRPKM using Ensemble gtf tracks. EdgeR (see, e.g., Robinson et al.Bioinformatics (2010) 26:139-40) was used to estimate significance ofdifferential expression between KO and parental samples. Overall geneexpression changes were considered significant if passed FDR<5%, Fold>3thresholds. ChIP-seq data was aligned using bowtie (see, e.g., Langmeadet al. Genome Biol. (2009) 10:R25) against hg19 version of the humangenome and HOMER (see, e.g., Heinz et al. Mol Cell (2010) 38:576-89) wasused to call significant peaks in parental vs. CARM1 knockout comparisonusing style histone option and peaks that passed FDR<1% threshold werecalled significantly decreased in knockout cells. Genes that hadsignificantly decreased in knockout EZH2 and H3K27Me3 peak wereconsidered and overlapped with genes significantly upregulated inknockout cells. Significance of overlap was tested using hypergeometrictest using 57,736 Ensemble genes as a population size. Gene setenrichment analysis of gene sets was done using QIAGEN's Ingenuity®Pathway Analysis software (IPA®, QIAGEN RedwoodCity,www.qiagen.com/ingenuity) using “Diseases & Functions” and“Upstream Analysis” options. Functions with at least 10 member genesthat passed p<10⁻⁵ threshold and upstream regulators (transcriptionfactors only) that passed p<10⁻¹⁰ and had a significantly predictedactivation state (|Z|>2) were considered. TCGA data RNASeqV2 level 3expression data for 579 ovarian cancer (OV) patients was downloaded andtested for negative association with CARM1 expression using bothSpearman and Pearson correlation and results overlapped with CARM1parental/KO data using Entrez gene ID. The RNA-seq and ChIP-seq data wassubmitted to the Gene Expression Omnibus (GEO) database and can beaccessed using accession number: GSE95645.

Statistical Analysis. Statistical analyses were performed using GraphPadPrism 5 (GraphPad) for Mac OS. Quantitative data are expressed asmean±S.E.M. unless otherwise stated. Spearman's or Pearson's test wasused to measure statistical correlation. For all statistical analyses,the level of significance was set at 0.05.

Results

CARM1 is Amplified in EOC and its Amplification/High ExpressionCorrelates with Poor Survival.

Analysis of high-throughput genetic profiles from The Cancer GenomicsAtlas (TCGA) revealed amplification of CARM1 in ˜10% of high-gradeserous ovarian carcinomas (HGSOC) (FIG. 14) (Cancer Genome AtlasResearch N. Nature (2011) 474:609-15). Consistently, CARM1 was expressedat a higher level in laser capture and microdissected HGSOCs comparedwith normal human ovarian surface epithelial (HOSE) cells (FIG. 15) (Moket al. Cancer Cell (2009) 16:521-532). In addition, recent evidenceindicates that the majority of HGSOC likely develops from the fallopiantube fimbriae epithelium (FTE) (see, e.g., Bowtell et al. Nat Rev Cancer(2015) 15:668-79 and Dubeau et al. Ann Oncol. (2013) 24 Suppl 8,viii28-viii35). Indeed, CARM1 was also expressed at a higher level inlaser capture and microdissected HGSOCs compared with normal humanfallopian tube epithelial (FTE) cells (FIG. 16) (Tone et al. Clin CancerRes. (2008) 14:4067-78). Likewise, CARM1 was expressed at higher levelsin a number of EOC cell lines compared with either FTE or HOSE cells(FIG. 17). Interestingly, CARM1 amplification and BRCA1/2 mutations donot typically occur in the same tumor (FIG. 14) (Cancer Genome AtlasResearch N. Nature (2011) 474:609-15). Consistent with its oncogenicrole in EOC, CARM1 amplification or high expression predicted a shorteroverall survival in TCGA HGSOC database and an independent EOC patientcohort based on gene expression profiling (FIGS. 18 and 19). Thus, CARM1is amplified in EOC, and its amplification/high expression correlateswith a poor overall survival in EOC patients.

Towards understanding the role of CARM1 in EOC, a CARM1 knockout (CARM1KO) clone was generated in CARM1-high A1847 cells using the CRISPRmethodology (FIG. 20). Consistent with CARM1's growth-promoting rolereported in other cancer types (Yang et al. Nat Rev Cancer (2013)13:37-50), CARM1 KO A1847 cells exhibited a decrease in growth comparedwith parental controls (FIG. 21). Similar observations were made withshRNA-mediated CARM1 knockdown in CARM1-high EOC cell lines such asOVCAR10 and A1847 (FIGS. 22-25). Thus, CARM1 inhibition suppresses thegrowth of EOC cells.

EZH2 Inhibitors Selectively Suppress the Growth of CARM1-High Cells.

CARM1 asymmetrically dimethylates substrates involved in epigenetic genetranscription (Yang et al. Nat Rev Cancer (2013) 13:37-50). Thissuggests that epigenetic mechanisms play a key role in mediating theoncogenic activity of CARM1. Thus, an unbiased evaluation was performedof a set of 23 small molecule epigenetic inhibitors (Bitler et al.Nature Medicine (2015) 21:231-8). Each individual inhibitor wasevaluated for its ability to selectively suppress the growth ofCARM1-expressing cells compared with CARM1 KO cells. Interestingly, bothof the EZH2 inhibitors in the set (namely GSK126 and UNC1999) showedselectivity against CARM1-expressing cells (FIGS. 26 and 27) (see, e.g.,McCabe et al. Nature (2012) 492:108-12 and Konze et al. ACS Chem Biol.(2013) 8: 1324-34). This is not due to a reduced proliferation of CARMKO cells because: (1) a number of small molecule inhibitors were equallyeffective in suppressing the growth of both CARM-expressing and KO cells(e.g., CI994, FIG. 28); and (2) the data was normalized to the growth ofvehicle-treated CARM1-expressing or KO cells to control for variation incell growth. The observed CARM1-dependent selectivity by EZH2 inhibitorswas not due to changes in EZH2 levels because its expression was notaltered and levels of its enzymatic product H3K27Me3 were not changed byCARM1 knockout (FIG. 29). However, EZH2 is overexpressed in CARM-highprimary HGSOC and cell lines compared to either HOSE cells or FTE cells(FIGS. 17, 30, and 31) (Cancer Genome Atlas Research N. Nature (2011)474: 609-15 and Tone et al. Clin Cancer Res. (2008) 14:4067-78).

These studies focus on GSK126. It was determined that 10 μM GSK126 wassufficient to inhibit >95% of the enzymatic activity of EZH2 asindicated by the decrease in H3K27Me3 levels (FIG. 32). Note that EZH2inhibition did not affect the expression of CARM1 (FIG. 32). GSK126 didnot affect EZH2 protein levels but instead selectively inhibited itsmethyltransferase activity as evidenced by a dose-dependent decrease inH3K27Me3 levels (FIG. 32). Thus, a concentration of 10 μM GSK126 wasused for subsequent studies. Validating the pharmacological screen,there was a correlation between CARM1 expression levels and cellularresponse to GSK126 in a panel of EOC cell lines (FIGS. 17, 33, and 34).Notably, GSK126 did not affect the growth of either HOSE or FTE cells(FIG. 33). Similar results were obtained in CARM1-high parental andCARM1 KO cells in both conventional 2D and 3D cultures using Matrigelextracellular matrix (FIGS. 35 and 36). EZH2 is expressed at comparablelevels in normal controls and CARM1-low EOC cells that did not respondto GSK126 such as CAOV3 and PEO1 (FIGS. 17 and 33), indicating that theobserved selectivity was not due to EZH2 upregulation alone. Consistentwith the observed growth inhibition, markers of apoptosis were inducedby EZH2 inhibition in a CARM1-dependent manner (e.g., FIGS. 37, 38, and39A). These data point to sensitivity to EZH2 inhibitors as a unique andexploitable therapeutic vulnerability in CARM1-high EOCs.

To limit the potential off-target effects and validate that the observedeffects were due to inhibition of EZH2's methyltransferase activity,genetic rescue experiments were performed. Indeed, apoptosis induced byEZH2 knockdown could be rescued by wild-type EZH2 but not by a mutantwith inactivated catalytic activity (FIGS. 40 and 41). Consistently, thecell growth inhibition induced by EZH2 knockdown was rescued bywild-type EZH2 but not by a catalytically inactive EZH2 mutant (FIGS. 42and 43). Evidence suggests that EZH2 inhibitor can also affect cellgrowth by destabilizing PRC2 complex in a catalytic activity-independentmanner. Notably, the EZH2 inhibitor GSK126 did not weaken theinteraction between PRC2 subunits EZH2 and SUZ12 (FIG. 39B), supportingthe notion that the observed selectivity against CARM1 was not due todestabilization of PRC2 complex. Together, and without being limited toany one theory, it has been concluded that the observed selectivityagainst CARM1 by EZH2 inhibitor is due to inhibition of itsmethyltransferase activity.

CARM1 Promotes the Silencing of EZH2 Target Tumor Suppressor Genes.

To explore the mechanistic basis of the selectivity against EZH2inhibitor, RNA-deep sequencing (RNA-seq) was performed in parentalcontrol and CARM1 KO cells (FIG. 44). To identify direct EZH2 targetgenes that are regulated by CARM1, EZH2 and H3K27Me3 chromatinimmunoprecipitation were performed followed by deep sequencing(ChIP-seq) analysis in control and CARM1 KO cells (FIG. 44). Since CARM1may promote EZH2-dependent gene silencing, the genomic loci were focusedupon that showed a decrease in association with EZH2/H3K27Me3 in CARM1KO cells compared with controls (e.g., FIGS. 44 and 45).Cross-referencing RNA-seq and ChIP-seq data (GEO access number:GSE95645) revealed a list of 218 direct EZH2/H3K27Me3 target genes thatwere downregulated by CARM1 (>3-fold) (FIGS. 44, 46, and 47). Thisrepresents a 8.3-fold enrichment of EZH2/H3K27Me3 target genes among the2084 genes upregulated at least 3-fold in CARM1 KO cells compared withparental controls (P=1.5×10⁻¹³⁶) (FIG. 48). Finally, the list of 218direct EZH2/H3K27Me3 target genes were cross-referenced with 528 TCGAhigh-grade serous ovarian carcinomas gene expression profiles andidentified genes that negatively correlated with CARM1 expression inthese cases (FIG. 44). These prioritizations led to a list of 36 EZH2direct target genes that negatively correlated with CARM1 expression(FIG. 44). Pathway analysis revealed that the top functional pathwayenriched in these genes was apoptosis (n=19, P=2.6×10⁻⁶) (FIGS. 44 and49). Notably, three of the ranked apoptosis-regulating genes (DAB2 (Heet al. J Biol Chem. (2001) 276:26814-8), DLC1 (Barras et al. CancerMetastasis Rev. (2014) 33:87-100 and Zhou et al. Oncogene (2004)23:1308-13) and NOXA (Ploner et al. Oncogene (2008) 27 Suppl 1: S84-92))were known tumor suppressors that are implicated in suppressingproliferation and promoting apoptosis (FIG. 45), i.e., the phenotypesobserved when CARM1 was knocked out or EZH2 activity was inhibited withGSK126 (see, e.g., FIGS. 15-21, 26, and 28-31).

In parallel, Ingenuity Pathway Analysis was performed for the upstreamtranscription factor that regulates the genes differentially expressedin parental control and CARM1 KO cells. The top upstream transcriptionregulator identified was the SMARCA4 (also known as BRG1), the catalyticsubunit of the SWI/SNF complex (P=1.22×10⁻³⁰) (FIGS. 50 and 51).Notably, inhibition of EZH2 activity is known to be synthetically lethalwith inactivation of the SWI/SNF complex due to antagonistic regulationof the same set of genes by the EZH2/PRC2 and the SWI/SNF complexes(Kadoch et al. Sci Adv. (2015) 1: e1500447). CARM1 methylates the R1064residue of BAF155, a core subunit of the SWI/SNF complex (Wang et al.Cancer Cell (2014) 25: 21-36). Indeed, the observed changes inselectivity against CARM1 KO or knockdown cells correlated with thedecrease in R1064 methylated BAF155 (BAF155Me) levels (see FIGS. 50, 52,and 53). However, EZH2 inhibition did not affect BAF155Me levels (FIG.54). Together, these data suggest that CARM1 may promote EZH2-mediatedsilencing by altering the antagonism between PRC2 and SWI/SNF complexvia BAF155Me.

CARM1 Regulates Antagonism between BAF155 and EZH2 by MethylatingBAF155.

A validation study was first performed to determine whether theidentified genes were regulated by CARM1 and EZH2 inhibitor GSK126.Indeed, the expression of the identified genes was upregulated by eitherEZH2 inhibition by GSK126 treatment or CARM1 knockout or knockdown(FIGS. 55 and 56). Notably, GSK126 treatment and CARM1 knockout did notappear to have additive effects on the expression of these genes (FIG.55), indicating that they probably function in the same pathway toregulate the expression of these genes. As a control, knownCARM1-regulated BAF155Me target genes such as the tumor suppressor TIMP3(Wang et al. Cancer Cell (2014) 25: 21-36) were downregulated by CARM KObut not by EZH2 inhibitor GSK126 (FIG. 57). This supports the notionthat CARM1 promotes the silencing of EZH2/BAF155 target genes butmediates the expression of CARM1-regulated BAF155Me targeted genes in anEZH2-independent manner. Our results also suggest that EZH2 does notregulate BAF155Me target genes.

ChIP analysis validated that the association of EZH2 and its enzymaticproduct H3K27Me3 with these gene loci was indeed CARM1-dependent (FIGS.58 and 59). EZH2 inhibitor decreased H3K27Me3 occupancy withoutaffecting EZH2's association with its target genes (FIGS. 58 and 59).Importantly, CARM1 KO led to loss of EZH2 from these target gene lociand a corresponding increase in the association of BAF155 with thesegene loci (FIG. 60). This result indicates that there is a switch fromEZH2 to unmethylated BAF155 in these gene loci when CARM1 is knockedout. Finally, the association of RNA polymerase II (Pol II) with thegene loci correlated with changes in their expression (FIGS. 55 and 61).In contrast, there was no significant enrichment of either EZH2 orH3K27Me3 in the promoter of the CARM1-regulated BAF155Me target genessuch as TIMP3 (Wang et al. Cancer Cell (2014) 25: 21-36) (FIGS. 62 and63). CARM1 KO but not EZH2 inhibitor GSK126 treatment decreased theassociation of BAF155 and Pol II with the TIMP3 promoter (FIGS. 64 and65). Since the antibody against BAF155Me was not suitable for ChIPanalysis, BAF155Me occupancy of target genes could not be directlyexamined. Nevertheless, the data support a model that CARM1 promotes thesilencing of EZH2/BAF155 target genes by displacing BAF155 viamethylation, which then permits the occupancy of the target genepromoters by EZH2 and their consequent repression by H3K27Me3 (FIG. 66).In contrast, CARM1 mediates the expression of CARM1-regulated BAF155Metargeted genes in an EZH2-independent manner (FIG. 66). Thus, CARM1regulates the antagonism between SWI/SNF and PRC2 through methylatingBAF155 (FIG. 66).

The effects of CARM1-mediated BAF155 methylation at the R1064 residue onthe expression of identified EZH2/BAF155 target genes were then testedto establish that the observed antagonism is BAF155Me dependent. Towardthis goal, in CARM1-high A1847 cells, endogenous BAF155 were replacedwith either mutant BAF155 that can no longer be methylated by CARM1(BAF155R1064K) or wild-type BAF155 (FIG. 67). Indeed, BAF155 R1064Kmutant but not wild-type BAF155 upregulated the expression of theEZH2/BAF155 target genes (FIG. 68), indicating that only theunmethylated BAF155 can be associated with these genes. This correlatedwith a decrease in EZH2 and its enzymatic product H3K27Me3 at thepromoter of these genes and a concurrent increase of BAF155'sassociation with these gene promoters (FIGS. 69 and 70). In contrast,the association of BAF155 with the BAF155Me target gene TIMP3 wasrescued by wild-type BAF155 but not the BAF155 R1064K, which correlatedwith the suppression of TIMP3 by BAF155 R1046K but not wild-type BAF155(FIGS. 72A and 72B). Together, these data further support the notionthat CARM1-mediated methylation of BAF155 drives a switch from BAF155 toEZH2 at the promoters of the BAF155/EZH2 target tumor suppressor genes(FIG. 66). Therefore, EZH2 inhibition reactivates the tumor suppressiveBAF155/EZH2 target genes to promote apoptosis and inhibit proliferationof CARM1-expressing cells (FIG. 66).

EZH2 Inhibitor GSK126 Suppresses the Growth of CARM1-Expressing EOC InVivo and Improves the Survival of Tumor Bearing Mice.

EZH2 inhibitors, such as GSK126, are used safely in clinical trials forhematopoietic malignancies (Ribrag et al. Eur J Cancer (2014) 50:197).To determine the effects of EZH2 inhibition in vivo on the growth ofCARM1-expressing ovarian tumors, two xenograft models were utilized. Inthe subcutenous xenograft models, the injected CARM1-expressing A1847cells were first allowed to grow for one week to establish the tumors.Mice were then randomized and treated daily with vehicle control orGSK126 (50 mg/kg) by intraperitioneal (i.p.) injection as reported (see,e.g., Bitler et al. Nature Medicine (2015) 21:231-8 and McCabe et al.Nature (2012) 492:108-12). Indeed, GSK126 treatment significantlyinhibited the growth of CARM1-expressing tumors (FIGS. 73 and 74). Incontrast, GSK126 treatment failed to inhibit the growth of tumors formedby CARM1 knockout A1847 cells (FIG. 75). To more closely mimic the tumormicroenvironment, A1847 cells were orthotopically transplanted into thebursa covering the ovary of immunocompromised NSG mice. Similarly, theinjected cells were first allowed to grow for one week to establish thetumors. Mice were then randomized and treated daily with vehicle controlor GSK126 (50 mg/kg) by intraperitioneal (i.p.) injection. Similar towhat was observed in subcutaneous xenograft models, the growth ofCARM1-expressing tumors was significantly inhibited by GSK126 in theorthotopic xenograft models (FIGS. 76 and 77). The survival of thetreated mice was followed after stopping the treatment regimens.Importantly, GSK126 significantly improved the survival of mice bearingthe orthotopically-transplanted CARM1-expressing tumors compared tocontrols (FIG. 78) (P=0.0023). Thus, the EZH2 inhibitor GSK126 was foundto significantly suppress the growth of CARM1-expressing tumors andimproved the survival of mice bearing these tumors.

The observed tumor growth suppression in vivo was correlated with themolecular pathways underlying the CARM1-dependent effects of EZH2inhibition. To do so, immunohistochemical (IHC) analysis was performedfor markers of cell proliferation (Ki67), apoptosis (cleaved caspase 3),H3K27Me3 and EZH2. H3K27Me3 staining was decreased by GSK126, whileGSK126 did not affect EZH2 staining (FIGS. 79 and 80). Further, GSK126treatment decreased the expression of Ki67 and increased the expressionof cleaved caspase 3 (FIGS. 79 and 80). Finally, the observed decreasein cell proliferation and increase in apoptosis correlated with theupregulation of the identified CARM1-regulated EZH2/BAF155 target genessuch as DAB2, DLC1 and NOXA by the EZH2 inhibitor GSK126 in vivo (FIG.81). In contrast, GSK126 did not affect the expression of Ki67 andcleaved caspase 3 in tumors formatted by CARM1 knockout cells despitethe reduction of H3K27Me3 by GSK126 (FIGS. 82 and 83). Likewise, GSK126did not increase the expression of the identified CARM1 regulatedEZH2/BAF155 target genes in tumors formed by CARM1 knockout cells (FIG.84). Together, these data support a model that EZH2 inhibitionsuppresses proliferation and promotes apoptosis in CARM1-expressingtumors by upregulating the EZH2/BAF155 target genes through regulatingthe antagonism between EZH2 (PRC2) and BAF155 (SWI/SNF) (FIG. 85).

Discussion

The foregoing data demonstrates a dependence of CARM1-expressing cellson EZH2 activity, reflecting the silencing of EZH2 target tumorsuppressor genes in a CARM1-dependent manner. Mechanistically, CARM1regulates the antagonism between EZH2 and BAF155 to drive the silencingof EZH2/BAF155 target tumor suppressor genes, which promotes apoptosisand inhibits proliferation. Specifically, CARM1-mediated methylation ofBAF155 leads to the switch from BAF155 to EZH2 at the promoters of theEZH2/BAF155 target genes. Indeed, inhibition of EZH2 activity byclinically applicable small molecule restored the expression of theEZH2/BAF155 target genes. Thus, CARM1 regulates the antagonism betweenthe BAF155-containing SWI/SNF complex and the EZH2-containing PRC2complex by methylating BAF155. In addition, CARM1-mediated methylationof BAF155 leads to the distribution of BAF155 to the BAF155Me targetgenes such as TIMP3 in an EZH2-independent manner. Without being limitedto any one theory, this suggests that CARM1 functions to promote theexpression of BAF155Me target genes while epigenetically silencingEZH2/BAF155 target genes through EZH2 mediated H3K27Me3.

CARM1 plays a context-dependent role in cancer. Whereas the prevailingdata support an overall oncogenic role of CARM1 in cancers, emergingevidence indicates that CARM1 may also positively regulate the activityof tumor suppressors (Wang et al. Mol Cell (2016) 64:673-87) and promotethe expression of tumor suppressor genes such as TIMP3 through BAF155Me(Wang et al. Cancer Cell (2014) 25:21-36). Thus, directly targetingCARM1 may have unintended tumor-promoting effects. In addition, CARM1 isspecifically required for postnatal survival (Yadav et al. Proc NatlAcad Sci USA (2003) 100:6464-8). Together, these caveats suggest thatdirectly targeting CARM1 may not be a valid therapeutic strategy. Incontrast, the foregoing data demonstrates that EZH2 inhibition cansuppress the growth of CARM1-expressing tumors and improves survival oftumor bearing mice. This correlates with reactivation of EZH2-mediatedsilencing of tumor suppressor genes implicated in promoting apoptosisand inhibiting proliferation. Thus, targeting EZH2 activity may beadvantageous compared to inhibition of CARM1 activity.

Analysis of HGSOC patients from TCGA revealed that CARM1 is amplified in˜10% and overexpressed in an additional ˜10% of spontaneous HGSOC. Incomparison, somatic BRCA1/2 mutations occur in ˜3-4% of these cases foreach gene that are among the most commonly mutated genes in HGSOC.Interestingly, CARM1 amplification does not typically occur in HGSOCwith mutations in BRCA1/2. Thus, there is an even greater need fordeveloping therapeutic approaches that correlate with CARM1 status. Thisis because platinum-based chemotherapy, the current standard of care,and emerging treatment with PARP inhibitors are typically more effectivein patients with BRCA1/2 inactivation. In view of the foregoing, CARM1overexpression and/or amplification may serve as a predictive marker forfurther development of EZH2 inhibitors as a therapy in treatingepithelial ovarian cancer.

In summary, this example demonstrates that targeting EZH2methyltrasferase activity through the use of EZH2 inhibitors inCARM1-expressing cells represents a viable therapeutic strategy.Notably, EZH2 inhibitors such as GSK126 are well-tolerated with limitedtoxicity in clinical trials for hematopoietic malignancies. Thus, thestudies described herein provide a scientific rationale for usingapplicable EZH2 inhibitors for CARM1-expressing cancers, such asCARM1-expressing ovarian cancers, for which novel therapeutics areurgently needed. Given that CARM1 overexpression is frequently observedin many different cancer types, the foregoing findings have far-reachingimplications for improving therapy for an array of cancer types.

1. A method of treating a cancer in a human subject suffering from thecancer in which cancer cells overexpress arginine methyltransferaseCARM1, the method comprising the step of administering a therapeuticallyeffective dose of an enhancer of zeste homolog 2 (EZH2) inhibitor to thehuman subject.
 2. The method of claim 1, wherein the cancer is ovariancancer.
 3. The method of claim 2, wherein the ovarian cancer isepithelial ovarian cancer.
 4. The method of claim 2, wherein theepithelial ovarian cancer is an epithelial ovarian tumor.
 5. The methodof claim 2, wherein the ovarian cancer is malignant ovarian cancer. 6.The method of claim 1, wherein the EZH2 inhibitor is selected from thegroup consisting of(S)-1-(sec-butyl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-methyl-6-(6-(piperazin-1-yl)pyridin-3-yl)-1H-indole-4-carboxamide(GSK126):

tazemetostat:

(R,Z)-1-(1-(1-(ethylsulfonyl)piperidin-4-yl)ethyl)-N-((2-hydroxy-4-methoxy-6-methylpyridin-3-yl)methyl)-2-methyl-1H-indole-3-carbimidicacid (CPI-169):

1-cyclopentyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(4-(morpholinomethyl)phenyl)-1H-indazole-4-carboxamide(EPZ-5687):

N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl((1R,4R)-4-((2-methoxyethyl)(methyl)amino)cyclohexyl)amino)-2-methyl-5-(3-morpholinoprop-1-yn-1-yl)benzamide(EPZ-11989):

1-isopropyl-N-((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-6-(2-(4-methylpiperazin-1-yl)pyridin-4-yl)-1H-indazole-4-carboxamide(GSK343):

N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-3-methyl-6-(6-(4-methylpiperazin-1-yl)pyridin-3-yl)-1H-indole-4-carboxamide(GSK503):

1-isopropyl-6-(6-(4-isopropylpiperazin-1-yl)pyridin-3-yl)-N-((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-1H-indazole-4-carboxamide(UNC-1999):

6-cyano-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(pentan-3-yl)-1H-indole-4-carboxamide(E11):

(1S,2R,5R)-5-(4-amino-1H-imidazo[4,5-c]pyridin-1-yl)-3-(hydroxymethyl)-3-cyclopentene-1,2-diol(DZNep):

sinefungin:

and pharmaceutically acceptable salts, solvates, hydrates, or cocrystalsthereof.
 7. The method of claim 1, wherein the overexpression ofarginine methyltransferase CARM1 is at a level selected from the groupof at least 2%, at least 5%, at least 8%, at least 10%, and at least 15%relative to a level in normal epithelial cells.
 8. The method of claim1, further comprising the step of administering a therapeuticallyeffective dose of a platinum drug to the human subject.
 9. The method ofclaim 8, wherein the platinum drug is selected from the group consistingof cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin,nedaplatin, triplatin tetranitrate, lipoplatin (liposomal cisplatin),and pharmaceutically acceptable salts, solvates, hydrates, cocrystals,or prodrugs thereof.
 10. A pharmaceutical composition for treating acancer in a human subject suffering from the cancer in which cancercells overexpress arginine methyl transferase CARM1, the pharmaceuticalcomposition comprising a therapeutically effective amount of an enhancerof zeste homolog 2 (EZH2) inhibitor or a pharmaceutically acceptablesalt, solvate, hydrate, cocrystal, or prodrug thereof, and apharmaceutically acceptable carrier.
 11. The pharmaceutical compositionof claim 10, wherein the EZH2 inhibitor is selected from the groupconsisting of(S)-1-(sec-butyl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-methyl-6-(6-(piperazin-1-yl)pyridin-3-yl)-1H-indole-4-carboxamide(GSK126):

tazemetostat:

(R,Z)-1-(1-(1-(ethylsulfonyl)piperidin-4-yl)ethyl)-N-((2-hydroxy-4-methoxy-6-methylpyridin-3-yl)methyl)-2-methyl-1H-indole-3-carbimidicacid (CPI-169):

1-cyclopentyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(4-(morpholinomethyl)phenyl)-1H-indazole-4-carboxamide(EPZ-5687):

N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl((1R,4R)-4-((2-methoxyethyl)(methyl)amino)cyclohexyl)amino)-2-methyl-5-(3-morpholinoprop-1-yn-1-yl)benzamide(EPZ-11989):

1-isopropyl-N-((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-6-(2-(4-methylpiperazin-1-yl)pyridin-4-yl)-1H-indazole-4-carboxamide(GSK343):

N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-3-methyl-6-(6-(4-methylpiperazin-1-yl)pyridin-3-yl)-1H-indole-4-carboxamide(GSK503):

1-isopropyl-6-(6-(4-isopropylpiperazin-1-yl)pyridin-3-yl)-N-((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-1H-indazole-4-carboxamide(UNC-1999):

6-cyano-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(pentan-3-yl)-1H-indole-4-carboxamide(E11):

(1S,2R,5R)-5-(4-amino-1H-imidazo[4,5-c]pyridin-1-yl)-3-(hydroxymethyl)-3-cyclopentene-1,2-diol(DZNep):

sinefungin:

and pharmaceutically acceptable salts, solvates, hydrates, cocrystals,or prodrugs thereof.
 12. The pharmaceutical composition of claim 10,comprising a therapeutically effective amount of a platinum drug or apharmaceutically acceptable salt, solvate, hydrate, or cocrystalthereof.
 13. The pharmaceutical composition of claim 10, wherein theplatinum drug is selected from the group consisting of cisplatin,carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatintetranitrate, lipoplatin (liposomal cisplatin), and pharmaceuticallyacceptable salts, solvates, hydrates, or cocrystal thereof.